Nicotiana Nucleic Acid Molecules and Uses Thereof

ABSTRACT

The present invention features  Nicotiana  nucleic acid sequences such as sequences encoding constitutive, or ethylene or senescence induced polypeptides, in particular cytochrome p450 enzymes, in  Nicotiana  plants and methods for using these nucleic acid sequences and plants to alter desirable traits, for example by using breeding protocols.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/665,451, filed Mar. 24, 2005, U.S. Provisional Application No.60/665,097, filed Mar. 24, 2005, U.S. Provisional Application No.60/646,764, filed Jan. 25, 2005, and U.S. Provisional Application No.60/607,357, filed Sep. 3, 2004, and U.S. Provisional Application No.60/566,235, filed Apr. 29, 2004.

The present application is also a divisional of U.S. application Ser.No. 11/116,881, filed Apr. 27, 2005, which is a continuation-in-partapplication of PCT/US2004/034065, filed Oct. 15, 2004, which claims thebenefit of U.S. Provisional Application No. 60/566,235, filed Apr. 29,2004, and which is a continuation-in-part of U.S. application Ser. No.10/934,944, filed Sep. 3, 2004. Further, this application is acontinuation-in-part of PCT/US2004/034218, filed Oct. 15, 2004, whichclaims the benefit of U.S. Provisional Application No. 60/566,235, filedApr. 29, 2004, and which is a continuation-in-part of U.S. applicationSer. No. 10/943,507, filed Sep. 17, 2004.

The present application is also a continuation-in-part of U.S.application Ser. No. 11/110,062, filed Apr. 19, 2005, which is acontinuation-in-part of U.S. application Ser. No. 10/934,944, filed Sep.3, 2004 and is a continuation-in-part of U.S. application Ser. No.10/943,507, filed Sep. 17, 2004. The entire contents of the foregoingare hereby incorporated by reference.

BACKGROUND

During tobacco ripening or curing the expression of various genes isaltered. Such genes may affect metabolic pathways involved in theformation of numerous secondary metabolites including terpenoids,polyphenols, and alkaloids that affect end-product quality traits. Forexample, the bioconversion of nicotine to form nornicotine during plantsenescence and in the post-harvest or leaf curing phase occurs in manyNicotiana species. Nicotine is the predominant source of nornicotine.The nornicotine alkaloid, is a substrate for microbe-mediatednitrosation to form the tobacco specific nitrosamine (TSNA)N′-nitrosonornicotine (NNN) during leaf curing or subsequent leafstorage and processing.

Genes expressed during tobacco ripening or curing may be constitutivelyexpressed, ethylene-induced or senescence-related genes, for instance,genes encoding a cytochrome p450. Cytochrome p450s, for example,catalyze enzymatic reactions for a diverse range of chemicallydissimilar substrates that include the oxidative, peroxidative, andreductive metabolism of endogenous and xenobiotic substrates. In plants,p450s participate in biochemical pathways that include the synthesis ofplant products such as phenylpropanoids, alkaloids, terpenoids, lipids,cyanogenic glycosides, and glucosinolates studied (Chappell, Annu Rev.Plant Physiol. Plant Mol. Biol. 46:521-547, 1995). Cytochrome p450s,also known as p450 heme-thiolate proteins, usually act as terminaloxidases in multi-component electron transfer chains, calledp450-containing monooxygenase systems. Specific reactions catalyzed bythese enzyme systems include demethylation, hydroxylation, epoxidation,N-oxidation, sulfooxidation, N-, S-, and O-dealkylations, desulfation,deamination, and reduction of azo, nitro, and N-oxide groups.

The diverse role of Nicotiana plant p450 enzymes has been implicated ineffecting a variety of plant metabolites such as phenylpropanoids,alkaloids, terpenoids, lipids, cyanogenic glycosides, glucosinolates,and a host of other chemical entities. Some p450 enzymes can impact thecomposition of plant metabolites. For example, it has been long desiredto improve the flavor and aroma of certain plants by altering a plant'sprofile of selected fatty acids through breeding; however very little isknown about mechanisms involved in controlling the levels of these leafconstituents. The down regulation or up regulation of p450 enzymesassociated with the modification of fatty acids may facilitateaccumulation of desired fatty acids that provide more preferred leafphenotypic qualities.

The function of p450 enzymes and their broadening roles in plantconstituents is still being discovered. For instance, a special class ofp450 enzymes was found to catalyze the breakdown of fatty acid intovolatile C6- and C9-aldehydes and β-alcohols that are major contributorsof “fresh green” odor of fruits and vegetables. The level of other noveltargeted p450s may be altered to enhance the qualities of leafconstituents by modifying lipid composition and related breakdownmetabolites in Nicotiana leaf. Several of these constituents in leaf areaffected by senescence that stimulates the maturation of leaf qualityproperties. Still other reports have shown that p450s enzymes are playafunctional role in altering fatty acids that are involved inplant-pathogen interactions and disease resistance.

The large multiplicity of p450 enzyme forms, their differing structureand function have made their research on Nicotiana p450 enzymes verydifficult before the present invention. In addition, cloning of p450enzymes has been hampered at least in part because thesemembrane-localized proteins are typically present in low abundance andoften unstable during purification. Hence, a need exists for theidentification of p450 enzymes in plants and the nucleic acid sequencesassociated with those p450 enzymes. In particular, only a few cytochromep450 proteins have been reported in Nicotiana. The inventions describedherein entail the discovery of cytochrome 450s and cytochrome p450fragments that correspond to several groups of p450 species based ontheir sequence identity.

In addition to the p450 sequences, the present invention encompasses thediscovery of other constitutive and ethylene or senescence inducedsequences that address the need for regulating metabolic pathwaysinvolved in the formation of secondary metabolites that affect thequality of a tobacco product. These sequences are also useful in thedevelopment of plant germplasms that have desirable traits for use inbreeding programs to develop more desirable germplasms, and especiallynon-GMO (genetically modified organism) type germplasms.

SUMMARY OF THE INVENTION

The present inventors have identified and characterized constitutive,and ethylene and senescence induced sequences, including a genomic cloneof nicotine demethylase, from tobacco. Also described herein is the useof these sequences in breeding methods and in methods to create a plant(e.g., a transgenic plant) having desirable traits, such as alteredlevels of nornicotine or N′-nitrosonornicotine (“NNN”) or both relativeto a control plant.

In one aspect, the invention features a breeding method for producing atobacco plant having decreased expression of a nicotine demethylasegene, the method including the steps of: (a) providing a first tobaccoplant having variant nicotine demethylase gene expression; (b) providinga second tobacco plant that contains at least one phenotypic trait; (c)crossing the first tobacco plant with the second tobacco plant toproduce an F1 progeny plant; (d) collecting seed of the F1 progeny forthe variant nicotine demethylase gene expression; and (e) germinatingthe seed to produce a tobacco plant having decreased expression of thenicotine demethylase gene.

In one embodiment, a tobacco plant is identified as variant for nicotinedemethylase gene expression (e.g., at the transcriptional, posttranscriptional, or translational levels or at the level of enzymaticactivity) using the sequences described herein and standard methodsknown in the art.

In another embodiment of this breeding method, the first tobacco plantincludes an endogenous nicotine demethylase gene having a mutation(e.g., a deletion, substitution, point mutation, translocation,inversion, duplication, or an insertion). In another embodiment, thefirst tobacco plant includes a nicotine demethylase gene having a nullmutation, includes a recombinant gene that silences an endogenousnicotine demethylase gene, or includes a nicotine demethylase havingreduced or altered enzymatic activity. In yet another embodiment, thenicotine demethylase gene of the first tobacco plant is absent. In stillanother embodiment, the first tobacco plant is a transgenic plant.

Exemplary first tobacco plants useful in the breeding methods disclosedherein include Nicotiana africana, Nicotiana amplexicaulis, Nicotianaarentsii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotianacorymbosa, Nicotiana debneyi, Nicotiana excelsior, Nicotiana exigua,Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotianahesperis, Nicotiana ingulba, Nicotiana knightiana, Nicotiana maritima,Nicotiana megalosiphon, Nicotiana miersii, Nicotiana nesophila,Nicotiana noctiflora, Nicotiana nudicaulis, Nicotiana otophora,Nicotiana palmeri, Nicotiana paniculata, Nicotiana petunioides,Nicotiana plumbaginifolia, Nicotiana repanda, Nicotiana rosulata,Nicotiana rotundifolia, Nicotiana rustica, Nicotiana setchelli,Nicotiana stocktonii, Nicotiana eastii, Nicotiana suaveolens orNicotiana trigonophylla. Desirably the first tobacco plant is Nicotianaamplexicaulis, Nicotiana benthamiana, Nicotiana bigelovii, Nicotianadebneyi, Nicotiana excelsior, Nicotiana glutinosa, Nicotianagoodspeedii, Nicotiana gossei, Nicotiana hesperis, Nicotiana knightiana,Nicotiana maritima, Nicotiana megalosiphon, Nicotiana nudicaulis,Nicotiana paniculata, Nicotiana plumbaginifolia, Nicotiana repanda,Nicotiana rustica, Nicotiana suaveolens or Nicotiana trigonophylla.Other first tobacco plants include varieties of Nicotiana tabacum (orNicotiana rustica) or transgenic lines associated therewith that havebeen engineered to have decreased levels of nicotine demethylase. Otherexemplary first tobacco plants include an Oriental, a dark tobacco, flueor air-cured tobacco, Virginia, or a Burley tobacco plant.

In another embodiment of the above-described breeding method, the secondtobacco plant is Nicotiana tabacum. Exemplary varieties of Nicotianatabacum include commercial varieties such as BU 64, CC 101, CC 200, CC27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176,Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL26H, GL 350, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358,K 394, K 399, K 730, KT 200, KY 10, KY 14, KY 160, KY 17, KY 171, KY907, KY 160, Little Crittenden, McNair 373, McNair 944, msKY 14×L8,Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC3, NC 4, NC 5, NC 6, NC 606, NC 71, NC 72, NC 810, NC BH 129, OXFORD207, ‘Perique’ tobacco, PVH03, PVH09, PVH19, PVHSO, PVH51, R 610, R 630,R 7-11, R 7-12, RG 17, RG 81, RG H4, RG H51, RGH 4, RGH 51, RS 1410, SP168, SP 172, SP 179, SP 210, SP 220, SP G-28, SP G-70, SP H20, SP NF3,TN 86, TN 90, TN 97, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, VA309, or VA 359.

In still other embodiments, the phenotypic trait of the second tobaccoplant includes disease resistance; high yield; high grade index;curability; curing quality; mechanical harvestability; holding ability;leaf quality; height, plant maturation (e.g., early maturing, early tomedium maturing, medium maturing, medium to late maturing, or latematuring); stalk size (e.g., a small, medium, or a large stalk); or leafnumber per plant (e.g., a small (e.g., 5-10 leaves), medium (e.g., 11-15leaves), or large (e.g., 16-21) number of leaves). In still otherembodiments, the method further includes self-pollinating or pollinatinga male sterile pollen acceptor, a pollen donor capable of being used inproduction of a hybrid or a male sterile hybrid with the plant of step(b) or backcrossing or self-pollinating plants produced from germinatedseed of step (e).

In another aspect, the invention features a method of breeding anicotine demethylase deficiency trait into a tobacco plant, the methodincluding the steps of: a) crossing a first tobacco plant having variantnicotine demethylase gene expression with a second tobacco plant; b)producing progeny tobacco plants of the cross; c) extracting a DNAsample from progeny tobacco plants; d) contacting the DNA sample with amarker nucleic acid molecule that hybridizes to a nicotine demethylasegene or fragment thereof; and e) performing a marker assisted breedingmethod for the variant nicotine demethylase gene expression trait. Forexample, plants are identified as having variant gene expression of anicotine demethylase and, if desired, are further tested for nicotinedemethylase gene expression or tested using standard alkaloid profilingor immunoblotting analysis. Typically such a marker assisted breedingmethod includes utilizing an amplified fragment length polymorphism,restriction fragment length polymorphism, random amplified polymorphismdisplay, single nucleotide polymorphism, a microsatellite marker, or atargeted induced local lesion in a tobacco genome.

In yet another aspect, the invention features a method of producingtobacco seed, including crossing anyone of the tobacco plants selectedfrom the group consisting of Nicotiana africana, Nicotianaamplexicaulis, Nicotiana arentsii, Nicotiana benthamiana, Nicotianabigelovii, Nicotiana corymbosa, Nicotiana debneyi, Nicotiana excelsior,Nicotiana exigua, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotianagossei, Nicotiana hesperis, Nicotiana ingulba, Nicotiana knightiana,Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiananesophila, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotianaotophora, Nicotiana palmeri, Nicotiana paniculata, Nicotianapetunioides, Nicotiana plumbaginifolia, Nicotiana repanda, Nicotianarosulata, Nicotiana rotundifolia, Nicotiana rustica, Nicotianasetchelli, Nicotiana stocktonii, Nicotiana eastii, Nicotiana suaveolens,and Nicotiana trigonophylla with itself. In one embodiment, the methodfurther includes a method of preparing hybrid tobacco seed, includingcrossing a tobacco plant having variant nicotine demethylase geneexpression to a second, distinct tobacco plant. In still anotherembodiment of this method, the crossing includes the steps of: (a)planting a seed of the cross resulting from the tobacco plant havingvariant nicotine demethylase gene expression and a second, distincttobacco plant; (b) growing tobacco plants from the seed until the plantsbear flowers; (c) pollinating a flower of the tobacco plant havingvariant nicotine demethylase gene expression with pollen from the secondtobacco plant or pollinating a flower of the second tobacco plant withpollen from the flower of the tobacco plant having variant nicotinedemethylase gene expression; and (d) harvesting seed resulting from thepollinating.

In still another aspect, the invention features a method for developinga tobacco plant in a tobacco breeding program including: (a) providing atobacco plant, or its components, having variant nicotine demethylasegene expression; and (b) employing the plant or plant components as asource of breeding material using tobacco plant breeding techniques.Exemplary plant breeding techniques useful for practicing this methodinclude bulk selection, backcrossing, self-pollination, introgression,pedigree selection, pureline selection, haploid/doubled haploidbreeding, or single seed descent.

In another aspect, the invention features a breeding method forproducing a tobacco plant having a modified attribute, the methodincluding the steps of: (a) providing a first tobacco plant having amodified attribute including variant gene expression of a nucleic acidmolecule selected from the group consisting of the nucleic acidsequences shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193; (b)providing a second tobacco plant that contains at least one phenotypictrait; (c) crossing the first tobacco plant with the second tobaccoplant to produce an F1 progeny plant; (d) collecting seed of the F1progeny for the modified attribute; and (e) germinating the seed toproduce a tobacco plant having the modified attribute. In oneembodiment, the first tobacco plant includes an endogenous nucleic acidmolecule selected from the group consisting of the nucleic acidsequences shown in FIGS. 2 to 5 and SEQ ID NOS: 446 to 2193, wherein thenucleic acid includes a mutation. Exemplary mutations include deletion,substitution, point mutation, translocation, inversion, duplication, oran insertion.

In still another embodiment, the first tobacco plant of theabove-described method includes an endogenous nucleic acid moleculeselected from the group consisting of the nucleic acid sequences shownin FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, wherein the nucleic acidincludes a null mutation. In still yet another embodiment, the firsttobacco plant includes a recombinant gene that silences expression ofendogenous nucleic acid molecule an endogenous nucleic acid moleculeselected from the group consisting of the nucleic acid sequences shownin FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193.

The first tobacco plant includes, if desired, an endogenous nucleic acidmolecule selected from the group consisting of the nucleic acidsequences shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, wherein thenucleic acid molecule encodes a polypeptide having reduced or alteredenzymatic activity. In still other embodiments, the first tobacco plantis a transgenic plant.

Exemplary first tobacco plants useful in the breeding methods disclosedherein include Nicotiana africana, Nicotiana amplexicaulis, Nicotianaarentsii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotianacorymbosa, Nicotiana debneyi, Nicotiana excelsior, Nicotiana exigua,Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotianahesperis, Nicotiana ingulba, Nicotiana knightiana, Nicotiana maritima,Nicotiana megalosiphon, Nicotiana miersii, Nicotiana nesophila,Nicotiana noctiflora, Nicotiana nudicaulis, Nicotiana otophora,Nicotiana palmeri, Nicotiana paniculata, Nicotiana petunioides,Nicotiana plumbaginifolia, Nicotiana repanda, Nicotiana rosulata,Nicotiana rotundifolia, Nicotiana rustica, Nicotiana setchelli,Nicotiana stocktonii, Nicotiana eastii, Nicotiana suaveolens orNicotiana trigonophylla. Other first tobacco plants include varieties ofNicotiana tabacum or Nicotiana rustica. Still other first tobacco plantis an Oriental, a dark tobacco, flue or air-cured tobacco, Virginia, ora Burley tobacco plant.

In another embodiment of the above-described breeding method, the secondtobacco plant is Nicotiana tabacum. Exemplary varieties of Nicotianatabacum include commercial varieties such as BU 64, CC 101, CC 200, CC27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176,Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL26H, GL 350, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358,K 394, K 399, K 730, KT 200, KY 10, KY 14, KY 160, KY 17, KY 171, KY907, KY 160, Little Crittenden, McNair 373, McNair 944, msKY 14×L8,Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC3, NC 4, NC 5, NC 6, NC 606, NC 71, NC 72, NC 810, NC BH 129, OXFORD207, ‘Perique’ tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 30 610, R630, R 7-11, R 7-12, RG17, RG 81, RGH4, RGH51, RGH 4, RGH 51, RS 1410,SP 168, SP 172, SP 179, SP 210, SP 220, SP G-28, SP G-70, SP H20, SPNF3, TN 86, TN 90, TN97, TN D94, TN D950, TR (Tom Rosson) Madole, VA309, VA 309, or VA 359.

In still other embodiments, the phenotypic trait of the second tobaccoplant includes disease resistance; high yield; high grade index;curability; curing quality; mechanical harvestability; holding ability;leaf quality; height, plant maturation (e.g., early maturing, early tomedium maturing, medium maturing, medium to late maturing, or latematuring); stalk size (e.g., a small, medium, or a large stalk); or leafnumber per plant (e.g., a small (e.g., 5-10 leaves), medium (e.g., 11-15leaves), or large (e.g., 16-21) number of leaves).

In still further embodiments, the method includes pollinating a malesterile or a male sterile hybrid with the plant of step (b) orbackcrossing or pollinating plants produced from germinated seed of step(e). In another aspect, the invention features a method of breeding anattribute into a tobacco plant, the method including the steps of: a)crossing a first tobacco plant having a modified attribute includingvariant gene expression of a nucleic acid molecule selected from thegroup consisting of the nucleic acid sequences shown in FIGS. 2 to 7 andSEQ ID NOS: 446 to 2193 with a second tobacco plant; b) producingprogeny tobacco plants of the cross; c) extracting a DNA sample fromprogeny tobacco plants; d) contacting the DNA sample with a markernucleic acid molecule that hybridizes to a nucleic acid moleculeselected from the group consisting of the nucleic acid sequences shownin FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193 or a fragment thereof; ande) performing a marker assisted breeding method for the modifiedattribute. Typically such a marker assisted breeding method includesutilizing an amplified fragment length polymorphism, restrictionfragment length polymorphism, random amplified polymorphism display,single nucleotide polymorphism, a microsatellite marker, or a targetedinduced local lesion in a tobacco genome.

In yet another aspect, the invention features a method of producingtobacco seed, including crossing anyone of the tobacco plants selectedfrom the group consisting of Nicotiana africana, Nicotianaamplexicaulis, Nicotiana arentsii, Nicotiana benthamiana, Nicotianabigelovii, Nicotiana corymbosa, Nicotiana debneyi, Nicotiana excelsior,Nicotiana exigua, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotianagossei, Nicotiana hesperis, Nicotiana ingulba, Nicotiana knightiana,Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiananesophila, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotianaotophora, Nicotiana palmeri, Nicotiana paniculata, Nicotianapetunioides, Nicotiana plumbaginifolia, Nicotiana repanda, Nicotianarosulata, Nicotiana rotundifolia, Nicotiana rustica, Nicotianasetchelli, Nicotiana stocktonii, Nicotiana eastii, Nicotiana suaveolensand Nicotiana trigonophylla with a tobacco plant having a modifiedattribute including variant gene expression of a nucleic acid moleculeselected from the group consisting of the nucleic acid sequences shownin FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193 to a second, distincttobacco plant.

In still another embodiment, crossing includes the steps of: (a)planting a seed of the cross resulting from the tobacco plant having themodified attribute and a second, distinct tobacco plant; (b) growingtobacco plants from the seed until the plants bear flowers; (c)pollinating a flower of the tobacco plant having a modified attributewith pollen from the second tobacco plant or pollinating a flower of thesecond tobacco plant with pollen from the plant of the tobacco planthaving the modified attribute; and (d) harvesting seed resulting fromthe pollinating.

In still another aspect, the invention features a method for developinga tobacco plant in a tobacco breeding program including: (a) providing atobacco plant, or its components, having a modified attribute includingvariant gene expression of a nucleic acid molecule selected from thegroup consisting of the nucleic acid sequences shown in FIGS. 2 to 7 andSEQ ID NOS: 446 to 2193; and (b) employing the plant or plant componentsas a source of breeding material using tobacco plant breedingtechniques. Exemplary plant breeding techniques include bulk selection,backcrossing, self-pollination, introgression, pedigree selection,pureline selection, haploid/doubled haploid breeding, or single seeddescent.

In related aspects the invention features a tobacco plant or componentsthereof, produced according to anyone of the aforementioned breedingmethods. In still a further related aspect, the invention features atissue culture of regenerable tobacco cells obtained from anyone of theplants bred or produced according to the methods described herein. Suchtissue cultures regenerate tobacco plants capable of expressing all thephysiological and morphological characteristics of the tobacco planthaving variant nicotine demethylase gene expression or a modifiedattribute. Exemplary regenerable cells are embryos, meristematic cells,seeds, pollen, leaves, roots, root tips, or flowers or are protoplastsor callus derived therefrom.

In still related aspects, the invention features a method of producing atobacco product involving: (a) providing a tobacco plant producedaccording to anyone of the aforementioned breeding methods; and (b)preparing a tobacco product from the tobacco plant. Exemplary tobaccoproducts include leaves or stems or both; a smokeless tobacco product; amoist or dry snuff; a chewing tobaccos; cigarette products; cigarproducts; cigarillos; pipe tobaccos; or bidis.

An aspect of the invention features an isolated genetic marker includinga nucleic acid sequence that is substantially identical, desirably atleast 70% identical, to a nucleic acid sequence shown in FIGS. 2 to 7and SEQ ID NOS: 446 to 2193. In a desirable embodiment of this aspect ofthe invention, the nucleic acid sequence includes a sequence thathybridizes under stringent conditions to the complement of a nucleicacid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193. Inother desirable embodiments, the nucleic acid sequence is constitutive,or ethylene or senescence induced. In addition, the nucleic acidsequence desirably encodes a polypeptide that is substantially identicalto an amino acid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to548.

In other desirable embodiments of the invention, the nucleic acidsequence is operably linked to a heterologous gene or the nucleic acidsequence is operably linked to an inducible, constitutive, pathogen- orwound-induced, environmentally- or developmentally-regulated, or cell-or tissue-specific promoter.

In another aspect, the invention features an expression vectorcontaining a nucleic acid sequence shown in FIGS. 2 to 7 and SEQ ID NOS:446 to 2193, where the vector is capable of directing expression of thepolypeptide encoded by the nucleic acid sequence.

Further aspects of the invention features a substantially purepolypeptide containing the amino acid sequence of a polypeptide shown inFIGS. 2 to 7 and SEQ ID NOS: 446 to 548, as well as antibodies thatspecifically recognize the polypeptide.

An additional aspect of the invention features a plant or plantcomponent containing an isolated nucleic acid sequence shown in FIGS. 2to 7 and SEQ ID NOS: 446 to 2193, where the nucleic acid sequence isexpressed in the plant or the plant component, or a nucleic acidsequence that encodes a polypeptide that is substantially identical toan amino acid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 548,where the nucleic acid sequence is expressed in the plant or the plantcomponent. Desirably, the plant or plant component is a species ofNicotiana, for example, a Nicotiana species shown in Table 8. In otherdesirable embodiments, the plant component is a leaf, e.g., a curedtobacco leaf, a stem, or a seed. A desirable embodiment features a plantfrom the germinated seed.

In a further aspect, the invention features a tobacco product containinga plant or plant component containing an isolated nucleic acid sequenceshown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, where the nucleicacid sequence is expressed in the plant or the plant component.Desirably, the expression of an endogenous gene in the cured tobaccoplant or plant component is silenced. In other desirable embodiments,the tobacco product is a smokeless tobacco product, moist or dry snuff,a chewing tobaccos, cigarette products, cigar products, cigarillos, pipetobaccos, or bidis.

In particular, the tobacco product of this aspect of the invention maycontain dark tobacco, milled tobacco, or include a flavoring component.

An additional aspect of the invention features a method for reducing theexpression or enzymatic activity of a constitutive, or an ethyleneinduced or senescence induced tobacco polypeptide in a plant cell. Thismethod involves reducing the level or enzymatic activity of anendogenous constitutive, or ethylene or senescence induced tobaccopolypeptide in the plant cell. In a desirable embodiment, the tobaccopolypeptide is a p450. In other desirable embodiments, the plant cell isfrom a species of Nicotiana, e.g., one of the Nicotiana species shown inTable 8.

In further desirable embodiments, reducing the level of the endogenousconstitutive, or ethylene or senescence induced tobacco polypeptideinvolves expressing a transgene encoding an antisense nucleic acidmolecule of a nucleic acid sequence shown in FIGS. 2 to 7 and SEQ IDNOS: 446 to 2193 or a nucleic acid sequence that encodes a polypeptidecontaining an amino acid sequence shown in FIGS. 2 to 7 and SEQ ID NOS:446 to 548 in the plant cell. In another desirable embodiment, thetransgene encodes a double-stranded RNA molecule of a constitutive, oran ethylene or senescence induced tobacco nucleic acid or amino acidsequence in the plant cell. In other desirable embodiments, thetransgene is expressed, for example, in a tissue-specific,cell-specific, or organ-specific manner. In addition, reducing the levelof the endogenous constitutive, or ethylene or senescence inducedtobacco polypeptide desirably involves co-suppression of theconstitutive, or ethylene or senescence induced tobacco polypeptide inthe plant cell. In another desirable embodiment, reducing the level ofthe endogenous constitutive, or ethylene or senescence induced tobaccopolypeptide involves expressing a dominant negative gene product in theplant cell. Desirably, the endogenous constitutive, or ethylene orsenescence induced tobacco polypeptide includes a mutation in a genethat encodes an amino acid sequence shown in FIGS. 2 to 7 and SEQ IDNOS: 446 to 548. In further desirable embodiments, reduced expressionoccurs at the transcriptional level, at the translational level, or atthe post-translational level.

An additional aspect the invention features a method for increasing theexpression or enzymatic activity of a constitutive, or an ethylene orsenescence induced tobacco polypeptide in a plant cell. This methodinvolves increasing the level or enzymatic activity of an endogenousconstitutive, or ethylene or senescence induced tobacco polypeptide inthe plant cell. In a desirable embodiment of this aspect of theinvention, the plant cell is from a species of Nicotiana, for example, aNicotiana species shown in Table 8. In another desirable embodiment,increasing the level of the constitutive, or ethylene or senescenceinduced tobacco polypeptide involves expressing a transgene including anucleic acid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193or a nucleic acid sequence that encodes a polypeptide containing anamino acid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 548 inthe plant cell. Desirably, increased expression occurs at thetranscriptional level, at the translational level, or at thepost-translational level.

A further aspect of the invention features a method of producing aconstitutive, or an ethylene or senescence induced tobacco polypeptide.This method involves the steps of: (a) providing a cell transformed withan isolated nucleic acid molecule containing a nucleic acid sequenceshown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, (b) culturing thetransformed cell under conditions for expressing the isolated nucleicacid molecule; and (c) recovering the constitutive, or ethylene orsenescence induced tobacco polypeptide. Desirably, the constitutive, orethylene or senescence induced tobacco polypeptide is a p450. In anotherdesirable embodiment, the invention features a recombinant constitutive,or ethylene or senescence induced tobacco polypeptide produced accordingto the method of this aspect of the invention.

In another aspect, the invention features a method of isolating aconstitutive, or an ethylene or senescence induced tobacco polypeptideor fragment thereof. This method involves the steps of: (a) contacting anucleic acid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193or a portion thereof with a nucleic acid preparation from a plant cellunder hybridization conditions providing detection of nucleic acidsequences having at least 70% or greater sequence identity to a nucleicacid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193; and (b)isolating the hybridizing nucleic acid sequences. Desirably, theconstitutive, or ethylene or senescence induced tobacco polypeptide is ap450.

A further aspect of the invention features an isolated nucleic acidmolecule, for example, a DNA sequence, containing a nucleotide sequenceencoding a nicotine demethylase. In desirable embodiments, thenucleotide sequence of the first aspect is substantially identical to anucleotide sequence encoding a tobacco nicotine demethylase, such as atobacco nicotine demethylase containing a nucleotide sequence that is atleast 70% identical to the nucleotide sequence of SEQ ID NO: 4 or SEQ IDNO: 5, or that contains nucleotides 2010-2949 and/or 3947-4562 of SEQ IDNO: 4, or that contains the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.The isolated nucleic acid molecule of the first aspect of the invention,for example, is operably linked to a promoter functional in a plant celland desirably is contained in an expression vector. In other desirableembodiments, the expression vector is contained in a cell, e.g., a plantcell. Desirably, the plant cell, such as a tobacco plant cell, isincluded in a plant. In another desirable embodiment, the inventionfeatures a seed, e.g., a tobacco seed, from a plant containing theexpression vector, where the seed includes an isolated nucleic acidmolecule that hybridizes under stringent conditions to the sequence ofSEQ ID NO: 4 operably linked to a heterologous promoter sequence.Furthermore, the invention features a plant derived from a germinatedseed containing the expression vector, a leaf, either green or cured,from the plant, and an article of manufacture made from the leaf.

In another desirable embodiment, the nucleotide sequence contains asequence that hybridizes under stringent conditions to the complement ofthe nucleotide sequence of SEQ ID NO: 4 and/or SEQ ID NO: 5, or to afragment of SEQ ID NO: 4 or SEQ ID NO: 5. Desirably, the nucleotidesequence encodes a nicotine demethylase that is substantially identicalto the amino acid sequence of SEQ ID NO: 3. In a further desirableembodiment of the first aspect of the invention, the nicotinedemethylase has at least 70% amino acid sequence identity to thenicotine demethylase amino acid sequence of SEQ ID NO: 3 or to afragment of a nicotine demethylase having altered (e.g., reduced)enzymatic activity as compared to the full-length polypeptide.Desirably, the nicotine demethylase includes the amino acid sequence ofSEQ ID NO: 3.

In another aspect, the invention features an isolated nucleic acidmolecule containing a promoter that hybridizes under stringentconditions to the sequence of SEQ ID NO: 8, or a fragment thereof thatdrives transcription. Desirably, the promoter (i) is induced followingtreatment with ethylene or during senescence; and (ii) includes (a) basepairs 1-2009 of SEQ ID NO: 4, or (b) at least 200 consecutive base pairsidentical to 200 consecutive base pairs of the sequence defined by basepairs 1-2009 of SEQ ID NO: 4, or (c) a 20 base pair nucleotide portionidentical in sequence to a 20 consecutive base pair portion of thesequence set forth in base pairs 1-2009 of SEQ ID NO: 4.

A further aspect of the invention features an isolated nucleic acidpromoter containing a nucleotide sequence having 50% or more sequenceidentity with the sequence of SEQ ID NO: 8. Desirably, this isolatednucleic acid promoter is induced following treatment with ethylene orduring senescence and, for example, includes the sequence of SEQ ID NO:8. Alternatively, the promoter may include a fragment obtainable fromSEQ ID NO: 8, where the fragment drives transcription of a heterologousgene or reduces or alters nicotine demethylase enzymatic activity (forexample, silences gene expression). In a desirable embodiment thepromoter sequence is operably linked to a heterologous nucleic acidsequence, and may, for example be contained in an expression vector. Inother desirable embodiments the expression vector is contained in acell, e.g., a plant cell. Desirably, the plant cell, such as a tobaccoplant cell, is included in a plant. In another desirable embodiment, theinvention features a seed, e.g., a tobacco seed, from a plant containingthe expression vector, where the seed includes an isolated nucleic acidmolecule that hybridizes under stringent conditions to the sequence ofSEQ ID NO: 8 operably linked to a heterologous nucleic acid sequence.Furthermore, the invention features a plant derived from a germinatedseed containing the promoter of this aspect of the invention, a leaf,either green or cured, from the plant, and an article of manufacturemade from the leaf.

Another aspect of the invention features a method of expressing aheterologous gene in a plant. This method involves (i) introducing intoa plant cell a vector containing a promoter sequence having 50% or moresequence identity with the sequence of SEQ ID NO: 8 operably linked to aheterologous nucleic acid sequence; and (ii) regenerating a plant fromthe cell. In addition, this method may involve sexually transmitting thevector to progeny and, further, may include the step of collecting theseed produced by the progeny.

In yet another aspect, the invention features a method of reducingexpression of nicotine demethylase in a tobacco plant. This methodincludes the steps of (i) introducing into the tobacco plant a vectorcontaining the sequence of SEQ ID NO: 8 or a fragment obtainable fromSEQ ID NO: 8 operably linked to a heterologous nucleic acid sequence and(ii) expressing the vector in the tobacco plant. In a desirableembodiment of this method, expression of the nicotine demethylase issilenced. In other desirable embodiment, the vector expresses RNA, suchas antisense RNA or an RNA molecule capable of inducing RNA interference(RNAi).

In a further desirable aspect, the invention features an isolatednucleic acid molecule containing an intron that hybridizes understringent conditions to the sequence of SEQ ID NO: 7, or a fragmentthereof that reduces or alters nicotine demethylase enzymatic activity(for example, silences gene expression) or can serve as a molecularmarker to identify nicotine demethylase nucleic acid sequences. In adesirable embodiment, the intron includes (a) base pairs 2950-3946 ofSEQ ID NO: 4, or (b) at least 200 consecutive base pairs identical to200 consecutive base pairs of the sequence defined by base pairs2950-3946 of SEQ ID NO: 4, or (c) a 20 base pair nucleotide portionidentical in sequence to a 20 consecutive base pair portion of thesequence set forth in base pairs 2950-3946 of SEQ ID NO: 4.

Another desirable aspect of the invention features an isolated nucleicacid intron including a nucleotide sequence having 50% or more sequenceidentity with the sequence of SEQ ID NO: 7, or a fragment thereof thatreduces or alters nicotine demethylase enzymatic activity (for example,silences gene expression) or can serve as a molecular marker to identifynicotine demethylase nucleic acid sequences. Silencing gene expressionmay, for example, involve homologous recombination (e.g., using thesequence of SEQ ID NO: 188, or a fragment thereof) or a mutation thatresults in a gene product that does not have nicotine demethylaseactivity. In particular, the intron may include the sequence of SEQ IDNO: 7 or a fragment obtainable from SEQ ID NO: 7. Desirably, an isolatednucleic acid molecule including an intron is operably linked to aheterologous nucleic acid sequence and this sequence desirably isincluded in an expression vector. In another embodiment, the expressionvector is contained in a cell, such as a plant cell. In particular, thecell may be a tobacco cell. A plant, e.g., a tobacco plant, including aplant cell plant containing the sequence of SEQ ID NO: 7 or a fragmentobtainable from SEQ ID NO: 7 operably linked to a heterologous nucleicacid sequence in an expression vector is another desirable embodiment ofthe present invention. Further, a seed, for example, a tobacco seed,from a plant, where the seed contains an intron that hybridizes understringent conditions to SEQ ID NO: 7 operably linked to a heterologousnucleic acid sequence is also desirable. Furthermore, the inventionfeatures a plant derived from the germinated seed containing the intronof this aspect of the invention, a leaf, either green or cured, from theplant, and an article of manufacture made from the green or cured leaf.

A further aspect of the invention features a method of expressing anintron in a plant. This method involves (i) introducing into a plantcell an expression vector containing the sequence of SEQ ID NO: 7 or afragment obtainable from SEQ ID NO: 7 operably linked to a heterologousnucleic acid sequence; and (ii) regenerating a plant from the cell. In adesirable embodiment, this method also involves (iii) sexuallytransmitting the vector to progeny, and may include the additional stepof collecting the seed produced by the progeny. The method desirablyincludes, for example, regenerating a plant from the germinated seed, aleaf, either green or cured, from the plant, and a method of making anarticle of manufacture from the leaf.

In yet another aspect, the invention features a method of reducingexpression of nicotine demethylase in a tobacco plant. This methodincludes the steps of (i) introducing into the tobacco plant a vectorcontaining the sequence of SEQ ID NO: 7 or a fragment obtainable fromSEQ ID NO: 7 operably linked to a heterologous nucleic acid sequence and(ii) expressing the vector in the tobacco plant. In a desirableembodiment of this method, expression of the nicotine demethylase issilenced. In other desirable embodiment, the vector expresses RNA, suchas antisense RNA or an RNA molecule capable of inducing RNA interference(RNAi).

In an additional aspect, the invention features an isolated nucleic acidmolecule containing an untranslated region that hybridizes understringent conditions to the sequence of SEQ ID NO: 9 or a fragmentthereof that can alter the expression pattern of a gene, reduces oralters nicotine demethylase enzymatic activity (for example, silencesgene expression), or can be used as a marker to identify nicotinedemethylase nucleic acid sequences. In a desirable embodiment of thisaspect of the invention, the untranslated region includes (a) base pairs4563-6347 of SEQ ID NO: 4, or (b) at least 200 consecutive base pairsidentical to 200 consecutive base pairs of the sequence defined by basepairs 4563-6347 of SEQ ID NO: 4, or (c) a 20 base pair nucleotideportion identical in sequence to a 20 consecutive base pair portion ofthe sequence set forth in base pairs 4563-6347 of SEQ ID NO: 4.

An additional desirable aspect of the invention features an isolatednucleic acid untranslated region containing a nucleotide sequence having50% or more sequence identity with the sequence of SEQ ID NO: 9.Desirably, the untranslated region includes the sequence of SEQ ID NO: 9or the untranslated region includes a fragment obtainable from SEQ IDNO: 9 that can alter the expression pattern of a gene, reduces or altersnicotine demethylase enzymatic activity (for example, silences geneexpression), or can be used as a marker to identify nicotine demethylasenucleic acid sequences. The untranslated region desirably is operablylinked to a heterologous nucleic acid sequence and may be contained inan expression vector. Further, this expression vector is desirablycontained in a cell, such as a plant cell, e.g., a tobacco cell. Anotherdesirable embodiment of the invention features a plant, such as atobacco plant, including a plant cell containing a vector that includesan isolated nucleic acid sequence that has 50% or more sequence identitywith the sequence of SEQ ID NO: 9 and is operably linked to aheterologous nucleic acid sequence.

The invention also features a seed; for example, a tobacco seed, from aplant, where the seed includes an untranslated region that hybridizesunder stringent conditions to SEQ ID NO: 9 operably linked to aheterologous nucleic acid sequence. Furthermore, the invention featuresa plant derived from a germinated seed containing the untranslatedregion of this aspect of the invention, a leaf, either green or cured,from the plant, and an article of manufacture made from the green orcured leaf.

In a further aspect, the invention features a method of expressing anuntranslated region in a plant. This method involves (i) introducinginto a plant cell a vector containing an isolated nucleic acid sequencethat has 50% or more sequence identity with the sequence of SEQ ID NO: 9and is operably linked to a heterologous nucleic acid sequence; and (ii)regenerating a plant from the cell. In addition, this method may alsoinvolve (iii) sexually transmitting the vector to progeny, anddesirably, includes the additional step of collecting the seed producedby the progeny. The method desirably includes regenerating a plant fromthe germinated seed, a leaf, either green or cured, from the plant, anda method of making an article of manufacture made from the green orcured leaf.

Furthermore, the invention features a method of reducing expression oraltering the enzymatic activity of nicotine demethylase in a tobaccoplant. This method includes the steps of (i) introducing into thetobacco plant a vector containing an isolated nucleic acid sequence thathas 50% or more sequence identity with the sequence of SEQ ID NO: 9 andis operably linked to a heterologous nucleic acid sequence and (ii)expressing the vector in the tobacco plant. Desirably, expression of thenicotine demethylase is silenced. In other desirable embodiments thevector expresses RNA, e.g., antisense RNA or an RNA molecule capable ofinducing RNA interference (RNAi).

Another aspect of the invention features an expression vector includinga nucleic acid molecule containing a nucleotide sequence encoding anicotine demethylase, where the vector is capable of directingexpression of the nicotine demethylase encoded by the isolated nucleicacid molecule. Desirably, the vector includes the sequence of SEQ ID NO:4 or SEQ ID NO: 5. In other desirable embodiments, the inventionfeatures a plant or plant component, e.g., a tobacco plant or plantcomponent (e.g., a tobacco leaf or stern), that includes a nucleic acidmolecule containing a nucleotide sequence encoding a polypeptide thatdemethylates nicotine.

A further aspect of the invention features a cell containing an isolatednucleic acid molecule that includes a nucleotide sequence encoding anicotine demethylase. Desirably this cell is a plant cell or a bacterialcell, such as an Agrobacterium.

Another aspect of the invention features a plant or plant component(e.g., a tobacco leaf or stem) containing an isolated nucleic acidmolecule that encodes a nicotine demethylase, where the nucleic acidmolecule is expressed in the plant or the plant component. Desirably,the plant or plant component is an angiosperm, a dicot, a solanaceousplant, or a species of Nicotiana. Other desirable embodiments of thisaspect are a seed or a cell from the plant or plant component, as wellas a leaf, either green or cured, derived from the plant and an articleof manufacture made therefrom.

In an additional aspect, the invention features a tobacco plant havingreduced expression of a nucleic acid sequence encoding a polypeptide,for example, one that includes the sequence of SEQ ID NO: 3, and thatdemethylates nicotine, where the reduced expression (or a reduction inenzymatic activity) reduces the level of nornicotine in the plant. In adesirable embodiment, the tobacco plant is a transgenic plant, such asone that includes a transgene that, when expressed in the transgenicplant, silences gene expression of an endogenous tobacco nicotinedemethylase.

In particular, the transgenic plant desirably includes one or more ofthe following: a transgene that expresses an antisense molecule of atobacco nicotine demethylase or an RNA molecule capable of inducing RNAinterference (RNAi); a transgene that, when expressed in the transgenicplant, co-suppresses expression of a tobacco nicotine demethylase; atransgene that encodes a dominant negative gene product, e.g., a mutatedform the amino acid sequence of SEQ ID NO: 3; a point mutation in a genethat encodes the amino acid sequence of SEQ ID NO: 3; a deletion in agene that encodes a tobacco nicotine demethylase; and an insertion in agene that encodes a tobacco nicotine demethylase.

In other desirable embodiments, reduced expression of a nucleic acidsequence encoding a polypeptide occurs at the transcriptional level, atthe translational level, or at the posttranslational level.

Another aspect of the invention features a tobacco plant containing arecombinant expression cassette stably integrated into the genomethereof, where the cassette is capable of effecting a reduction innicotine demethylase activity. Seeds of this tobacco plant are featuredin a desirable embodiment. Other desirable embodiments include leaf,either green or cured, derived from this plant and an article ofmanufacture made therefrom.

A further aspect of the invention features a method of expressing atobacco nicotine demethylase in a plant. This method involves (i)introducing into a plant cell an expression vector including a nucleicacid molecule containing a nucleotide sequence encoding a nicotinedemethylase; and (ii) regenerating a plant from the cell. In a desirableembodiment, this method features sexually transmitting the vector toprogeny, and desirably also includes the additional step of collectingthe seed produced by the progeny. Additional desirable embodimentsinclude a plant derived from the germinated seed, a leaf, either greenor cured, from the plant, and an article of manufacture made from thegreen or cured leaf.

An additional aspect of the invention features a substantially puretobacco nicotine demethylase. Desirably, this tobacco nicotinedemethylase includes an amino acid sequence having at least 70% identityto the amino acid sequence of SEQ ID NO: 3 or includes the amino acidsequence of SEQ ID NO: 3. In a desirable embodiment, the tobacconicotine demethylase, upon expression in a plant cell, converts nicotineto nornicotine. In other desirable embodiments, the tobacco nicotinedemethylase, upon expression in a plant cell, is predominantly localizedin leaves, or the tobacco nicotine demethylase is induced by ethylene oris expressed during plant senescence.

In a further aspect, the invention features a substantially pureantibody that specifically recognizes and binds to a tobacco nicotinedemethylase. Desirably, the antibody recognizes and binds to arecombinant tobacco nicotine demethylase, e.g., one containing thesequence of SEQ ID NO: 3 or a fragment thereof.

Another aspect of the invention features a method of producing a tobacconicotine demethylase. This method involves the steps of: (a) providing acell transformed with an isolated nucleic acid molecule containing anucleotide sequence encoding a polypeptide that demethylates nicotine;(b) culturing the transformed cell under conditions for expressing theisolated nucleic acid molecule; and (c) recovering the tobacco nicotinedemethylase. The invention also features a recombinant tobacco nicotinedemethylase produced according to this method.

In an additional aspect, the invention features a method of isolating atobacco nicotine demethylase or fragment thereof. This method involvesthe steps of: (a) contacting the nucleic acid molecule of SEQ ID NOS: 4,5, 7, 8, or 9 or a portion thereof with a nucleic acid preparation froma plant cell under hybridization conditions providing detection ofnucleic acid sequences having at least 70% or greater sequence identityto the nucleic acid sequence of SEQ ID NOS: 4, 5, 7, 8, or 9; and (b)isolating the hybridizing nucleic acid sequences.

In a further aspect, the invention features another method of isolatinga tobacco nicotine demethylase or fragment thereof. This method includesthe steps of: (a) providing a sample of plant cell DNA; (b) providing apair of oligonucleotides having sequence identity to a region of anucleic acid molecule having the sequence of SEQ ID NOS: 4, 5, 7, 8, or9; (c) contacting the pair of oligonucleotides with the plant cell DNAunder conditions suitable for polymerase chain reaction-mediated DNAamplification; and (d) isolating the amplified tobacco nicotinedemethylase or fragment thereof. In a desirable embodiment of thisaspect, the amplification step is carried out using a sample of cDNAprepared from a plant cell. In another desirable embodiment, the tobacconicotine demethylase encodes a polypeptide which is at least 70%identical to the amino acid sequence of SEQ ID NO: 3.

A further aspect of the invention features a method for reducing theexpression of tobacco nicotine demethylase in a plant or plantcomponent. This method involves the steps of: (a) introducing into plantcells a transgene encoding a tobacco nicotine demethylase operablylinked to a promoter functional in the plant cells to yield transformedplant cells; and (b) regenerating a plant or plant component from thetransformed plant cells, where the tobacco nicotine demethylase isexpressed in the cells of the plant or plant component, thereby reducingthe expression of tobacco nicotine demethylase in a plant or plantcomponent. In particular embodiments of this aspect of the invention,the transgene encoding the tobacco nicotine demethylase isconstitutively expressed or inducibly expressed, for example, in atissue-specific, cell-specific, or organ-specific manner. In anotherembodiment of this aspect of the invention, expression of the transgeneco-suppresses the expression of an endogenous tobacco nicotinedemethylase or any other polypeptide described herein.

A further aspect of the invention features another method for reducingthe expression of tobacco nicotine demethylase or any of the otherpolypeptides described herein in a plant or plant component. This methodincludes the steps of: (a) introducing into plant cells a transgeneencoding an antisense coding sequence of a tobacco nicotine demethylaseor an RNA molecule capable of inducing RNA interference (RNAi) operablylinked to a promoter functional in the plant cells to yield transformedplant cells; and (b) regenerating a plant or plant component from thetransformed plant cells, where the antisense or an RNA molecule capableof inducing RNA interference (RNAi) of the coding sequence of thetobacco nicotine demethylase is expressed in the cells of the plant orplant component, thereby reducing the expression of tobacco nicotinedemethylase in a plant or plant component. Desirably, the transgeneencoding an antisense sequence or an RNA molecule capable of inducingRNA interference (RNAi) of a tobacco nicotine demethylase isconstitutively expressed or is inducibly expressed, for instance in atissue-specific, cell-specific, or organ-specific manner. In otherdesirable embodiments the antisense or RNA molecule capable of inducingRNAi of the coding sequence of the tobacco nicotine demethylase containsthe complement of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 61, SEQ ID NO: 188, or afragment thereof.

An additional aspect of the invention features yet another method forreducing the expression of tobacco nicotine demethylase in a plant orplant component. This method involving the steps of: (a) introducinginto plant cells a transgene encoding a dominant negative gene productof a tobacco nicotine demethylase operably linked to a promoterfunctional in the plant cells to yield transformed plant cells; and (b)regenerating a plant or plant component from the transformed plantcells, where the dominant negative gene product of the tobacco nicotinedemethylase is expressed in the cells of the plant or plant component,thereby reducing the expression of tobacco nicotine demethylase in aplant or plant component. In particular embodiments of this aspect ofthe invention, the transgene encoding the dominant negative gene productis constitutively expressed or is inducibly expressed, for example, in atissue-specific, cell-specific, or organ-specific manner.

A further aspect of the invention features an additional method forreducing the expression or the enzymatic activity of tobacco nicotinedemethylase in plant cell. This method involves reducing the level of anendogenous tobacco nicotine demethylase, or its enzymatic activity, inthe plant cell. Desirably, the plant cell is from a dicot, a solanaceousplant, or a species of Nicotiana. In desirable embodiments of thisaspect, reducing the level of endogenous tobacco nicotine demethylaseinvolves expressing a transgene encoding an antisense nucleic acidmolecule or an RNA molecule capable of inducing RNA interference (RNAi)of a tobacco nicotine demethylase in the plant cell, or involvesexpressing a transgene encoding a double-stranded RNA molecule of atobacco nicotine demethylase in the plant cell. Desirably, thedouble-stranded RNA is an RNA sequence corresponding to the sequence ofSEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 61, SEQ ID NO: 188 or a fragment thereof. In anadditional embodiment, reducing the level of endogenous tobacco nicotinedemethylase involves co-suppression of the endogenous tobacco nicotinedemethylase in the plant cell or involves expressing a dominant negativegene product in the plant cell. In particular, the dominant negativegene product may include a gene that encodes a mutated form the aminoacid sequence of SEQ ID NO: 3 or any other amino acid sequence describedherein.

In other desirable embodiments of this aspect of the invention, theendogenous tobacco nicotine demethylase includes a point mutation in agene that encodes the amino acid sequence of SEQ ID NO: 3. In otherdesirable embodiments reducing the level of expression of an endogenoustobacco nicotine demethylase involves a deletion in a gene that encodesa tobacco nicotine demethylase or involves an insertion in a gene thatencodes a tobacco nicotine demethylase. The reduced expression may occurat the transcriptional level, at the translational level, or at thepost-translational level.

A further aspect of the invention features a method for identifying acompound which alters the expression of a tobacco nicotine demethylasein a cell. This method involves the steps of: (a) providing a cellcontaining a gene encoding a tobacco nicotine demethylase; (b) applyinga candidate compound to the cell; and (c) measuring expression of thegene encoding the tobacco nicotine demethylase, where an increase ordecrease in expression relative to an untreated control sample is anindication that the compound alters expression of the tobacco nicotinedemethylase.

In a desirable embodiment of this method, the gene of part (a) encodes atobacco nicotine demethylase having at least 70% identity to the aminoacid sequence of SEQ ID NO: 3. Desirably, the compound decreases orincreases expression of the gene that encodes the tobacco nicotinedemethylase.

In another aspect, the invention features another method for identifyinga compound which alters the activity of a tobacco nicotine demethylasein a cell. This method involves the steps of: (a) providing a cellexpressing a gene encoding a tobacco nicotine demethylase; (b) applyinga candidate compound to the cell; and (c) measuring the activity of thetobacco nicotine demethylase, where an increase or decrease in activityrelative to an untreated control sample is an indication that thecompound alters activity of the tobacco nicotine demethylase. In adesirable embodiment of this aspect of the invention, the gene of step(a) encodes a tobacco nicotine demethylase having at least 70% identityto the amino acid sequence of SEQ ID NO: 3. Desirably, the compounddecreases or increases the activity of the tobacco nicotine demethylase.

A further aspect of the invention features a cured tobacco plant orplant component containing (i) a reduced levels of nicotine demethylaseor (ii) a nicotine demethylase having an altered enzymatic activity anda reduced amount of a nitrosamine. Desirably, the plant component is atobacco leaf or tobacco stem. In a desirable embodiment, the nitrosamineis nornicotine, and the content of nornicotine desirably is less than 5mg/g, 4.5 mg/g, 4.0 mg/g, 3.5 mg/g, 3.0 mg/g, more desirably less than2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, more desirably less than 750μg/g, 500 μg/g, 250 μg/g, 100 μg/g, even more desirably less than 75μg/g, 50 μg/g, 25 μg/g, 10 μg/g, 7.0 μg/g, 5.0 μg/g, 4.0 μg/g, and evenmore desirably less than 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, 0.4 μg/g, 0.2μg/g, 0.1 μg/g, 0.05 μg/g, or 0.01 μg/g or wherein the percentage ofsecondary alkaloids relative to total alkaloid content therein is lessthan 90%, 70%, 50%, 30%, 10%, desirably less than 5%, 4%, 3%, 2%, 1.5%,1%, and more desirably less than 0.75%, 0.5%, 0.25%, or 0.1%. In anotherdesirable embodiment, the nitrosamine is N′-nitrosonomicotine (NNN), andthe content of N′—NNN desirably is less than 5 mg/g, 4.5 mg/g, 4.0 mg/g,3.5 mg/g, 3.0 mg/g, more desirably less than 2.5 mg/g, 2.0 mg/g, 1.5mg/g, 1.0 mg/g, more desirably less than 750 μg/g, 500 μg/g, 250 μg/g,100 μg/g, even more desirably less than 75 μg/g, 50 μg/g, 25 μg/g, 10μg/g, 7.0 μg/g, 5.0 μg/g, 4.0 μg/g, and even more desirably less than2.0 μg/g, 1.0 μg/g, 0.5 μg/g, 0.4 μg/g, 0.2 μg/g, 0.1 μg/g, 0.05 μg/g,or 0.01 μg/g or wherein the percentage of secondary alkaloids relativeto total alkaloid content contained therein is less than 90%, 70%, 50%,30%, 10%, desirably less than 5%, 4%, 3%, 2%, 1.5%, 1%, and moredesirably less than 0.75%, 0.5%, 0.25%, or 0.1%. In additional desirableembodiments of this aspect of the invention, the cured tobacco plant orplant component is a dark tobacco, Burley tobacco, flue cured tobacco,Virginia, air-cured tobacco, or Oriental tobacco.

Further, the cured tobacco plant or plant component of the inventiondesirably includes a recombinant nicotine demethylase gene, e.g., onecontaining the sequence of SEQ ID NO: 4 or. SEQ ID NO: 5, or a fragmentthereof. Desirably, the expression of an endogenous nicotine demethylasegene, or of any other nucleic acid sequence described herein, in thecured tobacco plant or plant component is silenced.

Another aspect of the invention features a tobacco product containing acured tobacco plant or plant component that includes (i) reducedexpression of a nicotine demethylase or any other polypeptide describedherein or (ii) a nicotine demethylase or another polypeptide describedherein having altered activity, and a reduced amount of a nitrosamine.Desirably, the tobacco product is smokeless tobacco, moist or dry snuff,a chewing tobaccos, cigarette products, cigar products, cigarillos, pipetobaccos, or bidis. In particular, the tobacco product of this aspect ofthe invention may contain dark tobacco, milled tobacco, or include aflavoring component.

The invention also features a method of making a tobacco product, e.g.,a smokeless tobacco product, containing (i) reduced expression of anicotine demethylase or (ii) a nicotine demethylase having altered(e.g., reduced) enzymatic activity, and a reduced amount of anitrosamine. This method involves providing a cured tobacco plant orplant component containing (i) a reduced level of nicotine demethylaseor (ii) a nicotine demethylase having an altered enzymatic activity anda reduced amount of a nitrosamine and preparing the tobacco product fromthe cured tobacco plant or plant component.

DEFINITIONS

“Enzymatic activity” is meant to include but is not limited todemethylation, hydroxylation, epoxidation, N-oxidation, sulfooxidation,N-, S-, and O-dealkylations, desulfation, deamination, and reduction ofazo, nitro, N-oxide, and other such enzymatically reactive chemicalgroups. Altered enzymatic activity refers to a decrease in enzymaticactivity (for example, of a tobacco nicotine demethylase) by at least10-20%, preferably by at least 25-50%, and more preferably by at least55-95% or greater relative to the activity of a control enzyme (forexample, a wild-type tobacco plant tobacco nicotine demethylase). Theactivity of an enzyme, such as a nicotine demethylase may be assayedusing methods standard in the art, for example, using the yeastmicrosome assays described herein.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, orsense or anti-sense, and unless otherwise limited, encompasses knownanalogues of natural nucleotides that hybridize to nucleic acids in amanner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence includes the complementarysequence thereof. The terms “operably linked,” “in operablecombination,” and “in operable order” refer to functional linkagebetween a nucleic acid expression control sequence (such as a promoter,signal sequence, or array of transcription factor binding sites) and asecond nucleic acid sequence, wherein the expression control sequenceaffects transcription and/or translation of the nucleic acidcorresponding to the second sequence. Desirably, an operably linkednucleic acid sequence refers to a fragment of a gene that is linked toother sequences of the same gene to form a full-length gene.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, expresses the nucleicacid or expresses a peptide, heterologous peptide, or protein encoded bya heterologous nucleic acid. Recombinant cells can express genes or genefragments in either the sense or antisense form or an RNA moleculecapable of inducing RNA interference (RNAi) that are not found withinthe native (nonrecombinant) form of the cell. Recombinant cells can alsoexpress genes that are found in the native form of the cell, but whereinthe genes are modified and re-introduced into the cell by artificialmeans.

A “structural gene” is that portion of a gene comprising a DNA segmentencoding a protein, polypeptide or a portion thereof, and excluding, forexample, the 5′ sequence which drives the initiation of transcription orthe 3′UTR. The structural gene may alternatively encode anontranslatable product. The structural gene may be one which isnormally found in the cell or one which is not normally found in thecell or cellular location wherein it is introduced, in which case it istermed a “heterologous gene.” A heterologous gene may be derived inwhole or in part from any source known to the art, including a bacterialgenome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNAor chemically synthesized DNA. A structural gene may contain one or moremodifications that could affect biological activity or itscharacteristics, the biological activity or the chemical structure ofthe expression product, the rate of expression or the manner ofexpression control. Such modifications include, but are not limited to,mutations, insertions, deletions and substitutions of one or morenucleotides.

The structural gene may constitute an uninterrupted coding sequence orit may include one or more introns, bounded by the appropriate splicejunctions. The structural gene may be translatable or non-translatable,including an antisense or an RNA molecule capable of inducing. RNAinterference (RNAi). The structural gene may be a composite of segmentsderived from a plurality of sources and from a plurality of genesequences (naturally occurring or synthetic, where synthetic refers toDNA that is chemically synthesized).

An “exon” as used herein in reference to a nucleic acid sequence ismeant a portion of the nucleic acid sequence of a gene, where thenucleic acid sequence of the exon encodes at least one amino acid of thegene product. An exon is typically adjacent to a noncoding DNA segmentsuch as an intron.

An “intron” as used herein in reference to a nucleic acid sequence ismeant a non-coding region of a gene that is flanked by coding regions.An intron is typically a noncoding region of a gene that is transcribedinto an RNA molecule but is then excised by RNA splicing duringproduction of the messenger RNA or other functional structural RNA.

A “3′UTR” as used herein in reference to a nucleic acid sequence ismeant a non-coding nucleic acid sequence proximal to a stop codon of anexon.

“Derived from” is used to mean taken, obtained, received, traced,replicated or descended from a source (chemical and/or biological). Aderivative may be produced by chemical or biological manipulation(including, but not limited to, substitution, addition, insertion,deletion, extraction, isolation, mutation, and replication) of theoriginal source.

“Chemically synthesized,” as related to a sequence of DNA, means thatportions of the component nucleotides were assembled in vitro. Manualchemical synthesis of DNA may be accomplished using well-establishedprocedures (Caruthers, Methodology of DNA and RNA Sequencing, (1983),Weissman (ed.), Praeger Publishers, New York, Chapter 1); automatedchemical synthesis can be performed using one of a number ofcommercially available machines.

Optimal alignment of sequences for comparison may be conducted, forexample, by the local homology algorithm of Smith and Waterman, Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.,1990) is available from several sources, including the National Centerfor Biological Information (NCBI, Bethesda, Md.) and on the internet,for use in connection with the sequence analysis programs blastp,blastn, blastx, tblastn, and tblastx. It can be accessed athttp://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determinesequence identity using this program is available athttp://www.ncbi.nlm.nih.gov/BLAST/blast help.html.

The terms “substantial amino acid identity” or “substantial amino acidsequence identity” as applied to amino acid sequences and as used hereindenote a characteristic of a polypeptide, wherein the peptide comprisesa sequence that has at least 70 percent sequence identity, preferably 80percent amino acid sequence identity, more preferably 90 percent aminoacid sequence identity, and most preferably at least 99 to 100 percentsequence identity as compared to the protein sequence shown in FIGS. 2to 7 and SEQ ID NOS: 446 to 548. Desirably, for a nicotine demethylase,sequence comparison is desirably compared for a region following thecytochrome p450 motif GXRXCX(G/A) (SEQ ID NO:2265) to the stop codon ofthe translated peptide.

The terms “substantial nucleic acid identity” or “substantial nucleicacid sequence identity” as applied to nucleic acid sequences and as usedherein denote a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 50 percent,preferably 60, 65, 70, or 75 percent sequence identity, more preferably81 or 91 percent nucleic acid sequence identity, and most preferably atleast 95, 99, or even 100 percent sequence identity as compared to areference group over region corresponding to the first nucleic acidfollowing the region encoding the cytochrome p450 motif GXRXCX(G/A) (SEQID NO:2265) to the stop codon of the translated peptide.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Stringent conditions are sequence dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. to about 20° C., usually about 10° C. to about 15° C.,lower than the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength and pH) at which 50% of the target sequence hybridizes toa matched probe. Typically, stringent conditions will be those in whichthe salt concentration is about 0.02 molar at pH 7 and the temperatureis at least about 60° C. For instance in a standard Southernhybridization procedure, stringent conditions will include an initialwash in 6×SSC at 42° C. followed by one or more additional washes in0.2×SSC at a temperature of at least about 55° C., typically about 60°C., and often about 65° C.

Nucleotide sequences are also substantially identical for purposes ofthis invention when said nucleotide sequences encode polypeptides and/orproteins which are substantially identical. Thus, where one nucleic acidsequence encodes essentially the same polypeptide as a second nucleicacid sequence, the two nucleic acid sequences are substantiallyidentical even if they would not hybridize under stringent conditionsdue to degeneracy permitted by the genetic code (see, Darnell et al.(1990) Molecular Cell Biology, Second Edition Scientific American BooksW.H. Freeman and Company New York for an explanation of codon degeneracyand the genetic code). Protein purity or homogeneity can be indicated bya number of means well known in the art, such as polyacrylamide gelelectrophoresis of a protein sample, followed by visualization uponstaining. For certain purposes high resolution may be needed and HPLC ora similar means for purification maybe used.

By an antibody that “specifically binds” or “specifically recognizes” aparticular polypeptide, such as a tobacco nicotine demethylase, is meantan increased affinity of the antibody for the polypeptide relative to anequal amount of any other protein. Desirable antibodies are antibodiesthat specifically bind a polypeptide having an amino acid sequence shownin FIGS. 2 to 7 and SEQ ID NOS: 446 to 548. For example, an antibodythat specifically binds to a tobacco nicotine demethylase containing theamino acid sequence of SEQ ID NO: 3 desirably has an affinity for itsantigen that is least 2-fold, 5-fold, 10-fold, 30-fold, or 100-foldgreater than for an equal amount of any other antigen, including relatedantigens. Binding of an antibody to an antigen, e.g., a tobacco nicotinedemethylase, may be determined by any number of standard methods in theart, e.g., Western analysis, ELISA, or co-immunoprecipitation.Antibodies that specifically bind a polypeptide, e.g., a nicotinedemethylase, are also useful for purifying the polypeptide.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) into a cell. A vector may act toreplicate DNA and may reproduce independently in a host cell. The term“vehicle” is sometimes used interchangeably with “vector.” The term“expression vector” as used herein refers to a recombinant DNA moleculecontaining a desired coding sequence and appropriate nucleic acidsequences necessary for the expression of the operably linked codingsequence in a particular host organism. Nucleic acid sequences necessaryfor expression in prokaryotes usually include a promoter, an operator(optional), and a ribosome binding site, often along with othersequences. Desirably, the promoter includes the sequence of SEQ ID NO:8, or a fragment thereof that drives transcription. Also desirable arepromoter sequences that have at least 50%, 60%, 75%, 80%, 90%, 95%, oreven 99% sequence identity to the sequence of SEQ ID NO: 8 and thatdrive transcription. Eucaryotic cells are known to utilize promoters,enhancers, and termination and polyadenylation signals, such as the3′UTR sequence of SEQ ID NO: 9. In some instances, it has been observedthat plant expression vectors require the presence of plant derivedintrons, such as the intron having the sequence of SEQ ID NO: 7, to havestable expression. As such, the sequence of SEQ ID NO: 7, or any otherintron having an appropriate RNA splice junction may be used as furtherdescribed herein. Desirable vectors include a nucleic acid sequencesshown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193.

For the purpose of regenerating complete genetically engineered plantswith roots, a nucleic acid may be inserted into plant cells, forexample, by any technique such as in vivo inoculation or by any of theknown in vitro tissue culture techniques to produce transformed plantcells that can be regenerated into complete plants. Thus, for example,the insertion into plant cells may be by in vitro inoculation bypathogenic or non-pathogenic A. tumefaciens. Other such tissue culturetechniques may also be employed.

“Plant tissue,” “plant component” or “plant cell” includesdifferentiated and undifferentiated tissues of plants, including, butnot limited to, roots, shoots, leaves, pollen, seeds, tumor tissue andvarious forms of cells in culture, such as single cells, protoplasts,embryos and callus tissue. The plant tissue may be in planta or inorgan, tissue or cell culture.

“Plant cell” as used herein includes plant cells in planta and plantcells and protoplasts in culture. “cDNA” or “complementary DNA”generally refers to a single stranded DNA molecule with a nucleotidesequence that is complementary to an unprocessed RNA molecule containingan intron, or a processed mRNA lacking introns. cDNA is formed by theaction of the enzyme reverse transcriptase on an RNA template.

“Tobacco” as used herein includes flue-cured, Virginia, Burley, dark,Oriental, and other types of plant within the genus Nicotiana. Seed ofthe genus Nicotiana is readily available commercially in the form ofNicotiana tabacum.

“Articles of manufacture” or “tobacco products” include products such asmoist and dry snuff, chewing tobaccos, cigarette products, cigarproducts, cigarillos, pipe tobaccos, bidis, and similar tobacco-derivedproducts.

By “gene silencing” is meant a decrease in the level of gene expression(for example, expression of a gene encoding a tobacco nicotinedemethylase) by at least 30-50%, preferably by at least 50-80%, and morepreferably by at least 80-95% or greater relative to the level in acontrol plant (for example, a wild-type tobacco plant). Reduction ofsuch expression levels may be accomplished by employing standard methodswhich are known in the art including, without limitation, RNAinterference, triple strand interference, ribozymes, homologousrecombination, virus-induced gene silencing, antisense andco-suppression technologies, expression of a dominant negative geneproduct, or through the generation of mutated genes using standardmutagenesis techniques, such as those described herein. Levels of atobacco nicotine demethylase polypeptide or transcript, or both, aremonitored according to any standard technique including, but not limitedto, Northern blotting, RNase protection, or immunoblotting.

By a “fragment” or “portion” of an amino acid sequence is meant at leaste.g., 20, 15, 30, 50, 75, 100, 250, 300, 400, or 500 contiguous aminoacids of any of the amino acid sequences shown in FIGS. 2 to 7 and SEQID NOS: 446 to 548. Exemplary desirable fragments are amino acids 1-313of the sequence of SEQ ID NO: 3 and amino acids 314-517 of the sequenceof SEQ ID NO: 3, as well as the sequence of SEQ ID NOS: 2 and 63. Inaddition, with respect to a fragment or portion of a nucleic acidsequence, desirable fragments include at least 100, 250, 500, 750, 1000,or 1500 contiguous nucleic acids of any of the nucleic acid sequencesshown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193. Exemplary desirablefragments are nucleic acids 1-2009, 2010-2949, 2950-3946, 3947-4562,4563-6347, and 4731-6347 of the sequence of SEQ ID NO: 4.

By a “substantially pure polypeptide” is meant a polypeptide that hasbeen separated from most components which naturally accompany it;however, other proteins found in the microsomal fraction associated witha preparation having an enzymatic activity of at least 8.3 pKat/mgprotein is also considered to be a substantially pure polypeptide.Typically, the polypeptide is substantially pure when it is at least60%, by weight, free from the proteins and naturally-occurring organicmolecules with which it is naturally associated. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight, the desirable polypeptide. Asubstantially pure polypeptide may be obtained, for example, byextraction from a natural source (for example, a tobacco plant cell); byexpression of a recombinant nucleic acid encoding the polypeptide; or bychemically synthesizing the protein. Purity can be measured by anyappropriate method, for example, column chromatography, polyacrylamidegel electrophoresis, or by HPLC analysis.

By “isolated nucleic acid molecule” is meant a nucleic acid sequencefree from the nucleic acid sequences that naturally flank the sequenceof the nucleic acid molecule in the genome of an organism.

By “transformed cell” is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, aDNA molecule, for example, a DNA molecule encoding a tobacco nicotinedemethylase or any of the nucleic acid sequences disclosed herein (e.g.,the nucleic acid sequences shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to2193). By a “tobacco nicotine demethylase” or “nicotine demethylase” asused herein, is meant a polypeptide that is substantially identical tothe sequence of SEQ ID NO: 3. Desirably, a tobacco nicotine demethylaseis capable of converting nicotine (C₁₀H₁₄N₂, also referred to as3-(1-methyl-2-pyrrolidinyl)pyridine) to nornicotine (C₉H₁₂N₂). Theactivity of a tobacco nicotine demethylase may be assayed using methodsstandard in the art, such as by measuring the demethylation ofradioactive nicotine by yeast-expressed microsomes, as described herein.

As provided herein, the terms “cytochrome p450” and “p450” are usedinterchangeably.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the genomic structure of the tobacconicotine demethylase gene.

FIG. 2 is an electrophoresis image showing PCR products of tobacco lineswith the Geno full-length (“FL”) primer set.

FIG. 3 is an electrophoresis image showing PCR products of tobacco lineswith primer sets (1), (2), (3), and (4) as stated in Example 17. Theapproximate sizes of the bands are 3,500 nucleotides (nt) for FL, 2,600nt for (1), 1,400 nt for (2), 600 nt for (3), and 1,400 nt for (4).

FIG. 4A shows a comparison of Sequence Groups.

FIG. 4B shows a comparison of Sequence Groups.

FIG. 4C shows a comparison of Sequence Groups.

FIG. 5A shows a comparison of Sequence Groups.

FIG. 5B shows a comparison of Sequence Groups.

FIG. 5C shows a comparison of Sequence Groups.

FIG. 5D shows a comparison of Sequence Groups.

FIG. 5E shows a comparison of Sequence Groups.

FIG. 5F shows a comparison of Sequence Groups.

FIG. 5G shows a comparison of Sequence Groups.

FIG. 5H shows a comparison of Sequence Groups.

FIG. 5I shows a comparison of Sequence Groups.

FIG. 5J shows a comparison of Sequence Groups.

FIG. 5K shows a comparison of Sequence Groups.

FIG. 6A shows a comparison of Sequence Groups.

FIG. 6B shows a comparison of Sequence Groups.

FIG. 6C shows a comparison of Sequence Groups.

FIG. 6D shows a comparison of Sequence Groups.

FIG. 6E shows a comparison of Sequence Groups.

FIG. 7 is a diagram showing the cloning of cytochrome p450 cDNAfragments by PCR. Primers used for the cloning are listed: DM (SEQ IDNO: 2255), DM4 (SEQ ID NO: 2256), DM12 (SEQ ID NO: 2257), DM13 (SEQ IDNO: 2258), DM17 (SEQ ID NO: 2259), OLIGO d(T) (SEQ ID NO: 2260), T7 (SEQID NO: 2261), and SP6 (SEQ ID NO: 2262).

DETAILED DESCRIPTION

Traditionally, numerous steps were involved in the development of anynovel, desirable plant germplasm. Plant breeding begins with theanalysis and definition of problems and weaknesses of the currentgermplasm, the establishment of program goals, and the definition ofspecific breeding objectives. The next step is selection of germplasmthat possess the traits to meet the program goals. The goal is tocombine in a single variety an improved combination of desirable traitsfrom the parental germplasm. Desirable traits include, for example,higher seed yield, resistance to diseases and insects, tolerance todrought and heat, and better agronomic quantities. However, theseprocesses, which lead to the final step of marketing and distribution,can take six to twelve years from the time the first cross is made.Accordingly, development of new varieties is a time-consuming processthat requires precise forward planning, efficient use of resources, anda minimum of changes in direction.

Improvement of plant varieties through genetic transformation has becomeincreasingly important for modem plant breeding. Genes of potentialcommercial interest, such as genes conferring specific, desired planttraits of disease resistance, insect resistance, or improved quality,may be incorporated into crop species through various gene transfertechnologies. The ability to manipulate gene expression provides a meansof producing new characteristics in transformed plants. In somesituations high or increased levels of gene expression may be desired.For example, it is desirable to increase production of a protein thatitself maximizes the disease resistance, yield, flavor, or any othercommercially desirable attribute of a plant. Similarly, the regulationof endogenous gene expression by, for example, gene silencing may resultin more valuable plants or plant products.

During tobacco ripening or curing, the activation, up-regulation, ordown-regulation of any of the genes identified as ethylene-induced orsenescence-related (e.g., those having the sequence of SEQ ID NOS:4, 40,44, 52, 54, 60, 70, 104, 138, 140, 158, 162, 188, 212, 226, 234, and288) may affect those metabolic pathways involved in the formation ofnumerous secondary metabolites including terpenoids, polyphenols,alkaloids, etc. that affect end-product quality traits (e.g., diseaseresistance, insect resistance, improved quality, modified aroma,modified flavor, and the like). Similarly affected by the genesidentified herein may be the metabolic pathways associated with the rateand type of dry matter accumulated during senescence or the partitioningof dry matter within the plant during senescence. Changes in the rateand type of starch accumulation, lignin formation, cellulose deposition,and sugar translocation could be demonstrated. The control of genesidentified herein may also affect those metabolic pathways involved indetermining senescence rates, the uniformity of senescence within a leafand among leaves of a single plant, and the induction of senescence byartificial or natural means. The senescence inducing agents oractivities that stimulate or activate the genes identified hereininclude, for example, chemicals such as dilute peroxides, pesticides,herbicides, growth regulators, heat treatments, wounding, or gases suchas ozone and elevated concentrations of carbon dioxide.

Identifying Tobacco Constitutively Expressed, or Ethylene or SenescenceInduced Sequences

In accordance with the present invention, RNA was extracted fromNicotiana tissue of converter and non-converter Nicotiana lines. Theextracted RNA was then used to create cDNA. Nucleic acid sequences ofthe present invention were then generated using two strategies.

In the first strategy, the poly A enriched RNA was extracted from planttissue and cDNA was made by reverse transcription PCR. The single strandcDNA was then used to create p450 specific PCR populations usingdegenerate primers plus a oligo d(T) reverse primer. The primer designwas based on the highly conserved motifs of other plant cytochrome p450gene sequences. Examples of specific degenerate primers are set forth inFIG. 1 of the US 2004/0103449 A1, US 2004/0111759 A1, and US2004/0117869 A1 patent application publications, which are herebyincorporated by reference. The sequence of fragments from plasmidscontaining appropriate size inserts was further analyzed. These sizeinserts typically ranged from about 300 to about 800 nucleotidesdepending on which primers were used.

In a second strategy, a cDNA library was initially constructed. The cDNAin the plasmids was used to create p450 specific peR populations usingdegenerate primers plus T7 primer on plasmid as reverse primer. As inthe first strategy, the sequence of fragments from plasmids containingappropriate size inserts was further analyzed.

Nicotiana plant lines known to produce high levels of nornicotine(converter) and plant lines having low levels of nornicotine may be usedas starting materials. Leaves can then be removed from plants andtreated with ethylene to activate p450 enzymatic activities definedherein. Total RNA is extracted using techniques known in the art. cDNAfragments can then be generated using PCR(RT-PCR) with the oligo d(T)primer (SEQ ID NO: 2260) as described in FIG. 161. The cDNA library canthen be constructed as more fully described in examples herein.

The conserved region of p450 type enzymes was used as a template fordegenerate primers, examples of which are shown in FIG. 161. Usingdegenerate primers, p450 specific bands were amplified by PCR. Bandsindicative for p450-like enzymes were identified by DNA sequencing. PCRfragments were characterized using BLAST search, alignment or othertools to identify appropriate candidates.

Sequence information from identified fragments was used to develop PCRprimers. These primers in combination with plasmid primers in cDNAlibrary were used to clone full length p450 genes. Large-scale Southernreverse analysis was conducted to examine the differential expressionfor all fragment clones obtained and in some cases full-length clones.In this aspect of the invention, these large-scale reverse Southernassays can be conducted using labeled total cDNAs from different tissuesas a probe to hybridize with cloned DNA fragments in order to screen allcloned inserts. Nonradioactive and radioactive (p32) Northern blottingassays were also used to characterize cloned p450 fragments andfull-length clones.

Once plant cells expressing the desired level of p450 enzyme areobtained, plant tissues and whole plants can be regenerated therefromusing methods and techniques well-known in the art. The regeneratedplants are then reproduced by conventional means and the introducedgenes can be transferred to other strains and cultivars by conventionalplant breeding techniques.

Ethylene-induced or senescence-related genes, for example, thoseidentified in SEQ ID NOS:4, 40, 44, 52, 54, 60, 70, 104, 138, 140, 158,162, 188, 212, 226, 234, and 288, may encode enzymes that are importantdeterminants of tobacco leaf quality parameters important for a varietyof tobacco products. The tobacco products include moist or dry snuff,chewing tobaccos, cigarettes, cigars, cigarillos, pipe tobaccos, bidis,and similar smoking products. The leaf quality parameters may include:visual attributes such as color, surface uniformity, texture, orvariegation; structural or physical characteristics as exemplified bylamina-to-stem ratio, oiliness, cigarette filling potential, bulkdensity, moisture retention, and pliability; chemical or biochemicaltraits related to flavor, aroma, fermentation capability, burn rates,burn temperatures, artificial flavor absorption and release; andgeneration of smoke constituents including tar or particulate matter,alkaloids, and other similar attributes. The enzymatic reactionsresulting from these ethylene-induced or senescence-related genes mayalso produce secondary metabolites influencing pathogen or insectinteractions that affect tobacco leaf yield and quality. For example,Wagner, et al. (Nature Biotechnology, 19:371-374, 2001) showed thatsuppression of a p450 hydroxylase gene greatly increases theaccumulation of cembratiene-ol, a secondary metabolite influencing aphidresistance.

Generation of Antibodies

Peptide specific antibodies were made by deriving their amino acidsequence and selecting peptide regions that were antigenic and uniquerelative to other clones. Rabbit antibodies were made to syntheticpeptides conjugated to a carrier protein. Western blotting analyses orother immunological methods were performed on plant tissue using theseantibodies. In addition, peptide specific antibodies were made forseveral full-length clones by deriving their amino acid sequence andselecting peptide regions that were potentially antigenic and wereunique relative to other clones. Rabbit antibodies were made tosynthetic peptides conjugated to a carrier protein. Western blottinganalyses were performed using these antibodies.

Downregulating Gene Expression and Altering Enzymatic Activity

Plants having decreased expression of a polypeptide are generatedaccording to standard gene silencing methods. (For reviews, see Arndtand Rank, Genome 40:785-797, 1997; Turner and Schuch, Journal ofChemical Technology and Biotechnology 75:869-882, 2000; and Klink andWolniak, Journal of Plant Growth Regulation 19(4):371-384, 2000.) Inparticular, tobacco nicotine demethylase nucleic acid sequences (e.g.,SEQ ID NOS:4, 5, 7, 8, and 9, or fragments thereof such as the sequenceof SEQ ID NOS: 1 and 62), as well as substantially identical nucleicacid sequences (e.g., the sequence of SEQ ID NO: 188) can be used toalter tobacco phenotypes or tobacco metabolites, for example,nornicotine in any Nicotiana species. Decreased expression of a tobacconicotine demethylase gene may be achieved using, for example, RNAinterference (RNAi) (Smith et al., Nature 407:319-320, 2000; Fire etal., Nature 391:306-311, 1998; Waterhouse et al., PNAS 95: 13959-13964,1998; Stalberg et al., Plant Molecular Biology 23:671-683, 1993;Brignetti et al., EMBO J. 17:6739-6746, 1998; Allen et al., NatureBiotechnology 22:1559-1566, 2004); virus-induced gene silencing (“VIGS”)(Baulcombe, Current Opinions in Plant Biology, 2:109-113, 1999; Cogoniand Macino, Genes Dev 10: 638-643, 2000; Ngelbrecht et al., PNAS 91:10502-10506, 1994); silencing the target gene by transferring a plantendogenous gene in the sense orientation (Jorgensen et al., Plant MolBiol 31: 957-973, 1996); expression of antisense gene; homologousrecombination (Ohl et al., Homologous Recombination and Gene Silencingin Plants. Kluwer, Dordrecht, The Netherlands, 1994); Cre/lox systems(Qin et al., PNAS 91:1706-1710, 1994; Koshinsky et al., The PlantJournal 23: 715-722, 2000; Chou, et al., Plant and Animal Genome VIIConference Abstracts, San Diego, Calif., 17-21 January, 1999); genetrapping and T-DNA tagging (Burns et al., Genes Dev. 8: 1087-1105, 1994;Spradling, et al., PNAS 92:10824; 10830, 1995; Skames et al.,BiolTechnology 8, 827-831, 1990; Sundaresan, et al., Genes Dev. 9:1797-1810, 1995); and any of the other possible gene silencing systemsthat are available in the science areas that result in the downregulation of expression of a tobacco polypeptide or in a reduction inits enzymatic activity. As further provided herein, any of the nucleicacid sequences provided herein may be down regulated or up regulatedusing techniques described herein and other technologies found in theart. Exemplary methods are described in more detail below.

RNA Interference

RNA interference (“RNAi”) is a generally applicable process for inducingpotent and specific post-translational gene silencing in many organismsincluding plants (see, e.g., Bosher et al., Nat. Cell Biol. 2:E31-36,2000; and Tavemarakis et al., Nat. Genetics 24:180-183, 2000). RNAiinvolves introduction of RNA with partial or fully double-strandedcharacter into the cell or into the extracellular environment.Inhibition is specific in that a nucleotide sequence from a portion ofthe target gene (e.g., a tobacco nicotine demethylase) is chosen toproduce inhibitory RNA. The chosen portion generally encompasses exonsof the target gene, but the chosen portion may also include untranslatedregions (UTRs), as well as introns (e.g., the sequence of SEQ ID NO: 7,or a nucleic acid sequence from a desired plant gene, such as anynucleic acid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to2193).

For example, to construct transformation vectors that produce RNAscapable of duplex formation, two nucleic acid sequences, one in thesense and the other in the antisense orientation, may be operablylinked, and placed under the control of a strong viral promoter, such asCaMV 5 358 or the promoter isolated from cassaya brown streak virus(CBSV). However, use of the endogenous promoter, such as the nicotinedemethylase promoter having the sequence of SEQ ID NO: 8, or a fragmentthereof that drives transcription, may also be desirable. The length ofthe tobacco nicotine demethylase nucleic acid sequences included in sucha construct is desirably at least 25 nucleotides, but may encompass asequence that includes up to the full-length tobacco nicotinedemethylase gene.

Constructs that produce RNAs capable of duplex formation may beintroduced into the genome of a plant, such as a tobacco plant, byAgrobacterium-mediated. transformation (Chuang et al., Proc. Natl. Acad.Sci. USA 97:4985-4990, 2000), causing specific and heritable geneticinterference in a tobacco nicotine demethylase. The double-stranded RNAmay also be directly introduced into the cell (i.e., intracellularly) orintroduced extracellularly, for example, by bathing a seed, seedling, orplant in a solution containing the double-stranded RNA.

Depending on the dose of double-stranded RNA material delivered, theRNAi may provide partial or complete loss of function for the targetgene. A reduction or loss of gene expression in at least 99% of targetedcells may be obtained. In general, lower doses of injected material andlonger times after administration of dsRNA result in inhibition in asmaller fraction of cells.

The RNA used in RNAi may comprise one or more strands of polymerizedribonucleotide; it may include modifications to either thephosphate-sugar backbone or the nucleoside. The double-strandedstructure may be formed by a single self-complementary RNA strand or bytwo complementary RNA strands and RNA duplex formation may be initiatedeither inside or outside the cell. The RNA may be introduced in anamount which allows delivery of at least one copy per cell. However,higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) ofdouble-stranded material may yield more effective inhibition Inhibitionis sequence specific in that nucleotide sequences corresponding to theduplex region of the RNA are targeted for genetic inhibition. RNAcontaining a nucleotide sequences identical to a portion of the targetgene is preferred for inhibition. RNA sequences with insertions,deletions, and single point mutations relative to the target sequencemay also be effective for inhibition. Thus, sequence identity may beoptimized by alignment algorithms known in the art and calculating thepercent difference between the nucleotide sequences. Alternatively, theduplex region of the RNA may be defined functionally as a nucleotidesequence that is capable of hybridizing with a portion of the targetgene transcript.

In addition, the RNA used for RNAi may be synthesized either in vivo orin vitro. For example, endogenous RNA polymerase in the cell may mediatetranscription in vivo, or cloned RNA polymerase can be used fortranscription in vivo or in vitro. For transcription from a transgene invivo or an expression construct, a regulatory region may be used totranscribe the RNA strand (or strands).

Triple Strand Interference

Endogenous tobacco nicotine demethylase gene expression or expression ofa nucleic acid fragment from a desired plant gene, such as any nucleicacid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, mayalso be downregulated by targeting deoxyribonucleotide sequencescomplementary to the regulatory region of a tobacco gene (e.g., promoteror enhancer regions) to form triple helical structures that preventtranscription of the tobacco gene in target cells. (See generally,Helene, Anticancer Drug Des. 6:569-584, 1991; Helene et al., Ann. N.Y.Acad. Sci. 660:27-36, 1992; and Maher, Bioassays 14:807-815, 1992.)

Nucleic acid molecules used in triple helix formation for the inhibitionof transcription are preferably single stranded and composed ofdeoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example; containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a “switchback” nucleic acidmolecule. Switchback molecules are synthesized in an alternating5′-3′,3′-5′ manner, such that they base pair with first one strand of aduplex and then the other, eliminating the necessity for a sizablestretch of either purines or pyrimidines to be present on one strand ofa duplex.

Ribozymes

Ribozymes are RNA molecules that act as enzymes and can be engineered tocleave other RNA molecules. A ribozyme may be designed to specificallypair with virtually any target RNA and cleave the phosphodiesterbackbone at a specific location, thereby functionally inactivating thetarget RNA. The ribozyme itself is not consumed in this process and canact catalytically to cleave multiple copies of mRNA target molecules.Accordingly, ribozymes may also be used as a means to down-regulateexpression of a tobacco nicotine demethylase. The design and use oftarget RNA-specific ribozymes is described in Haseloff et al. (Nature334:585-591, 1988). Preferably, the ribozyme includes at least about 20continuous nucleotides complementary to the target sequence (e.g., atobacco nicotine demethylase or a nucleic acid fragment from a desiredplant gene, such as any nucleic acid sequence shown in FIGS. 2 to 7 andSEQ ID NOS: 446 to 2193) on each side of the active site of theribozyme.

In addition, ribozyme sequences may also be included within an antisenseRNA to confer RNA-cleaving activity upon the antisense RNA and, thereby,increasing the effectiveness of the antisense construct.

Homologous Recombination

Gene replacement technology is another desirable method fordown-regulating expression of a given gene. Gene replacement technologyis based upon homologous recombination (see, Schnable et al., CurroOpinions Plant Biol. 1:123-129, 1998). The nucleic acid sequence of theenzyme of interest such as a tobacco nicotine demethylase or apolypeptide encoded by any nucleic acid sequence shown in FIGS. 2 to 7and SEQ ID NOS: 446 to 2193 can be manipulated by mutagenesis (e.g.,insertions, deletions, duplications or replacements) to decreaseenzymatic function. The altered sequence can then be introduced into thegenome to replace the existing, e.g., wild-type, gene via homologousrecombination (Puchta et al., Proc. Natl. Acad. Sci. USA 93:5055-5060,1996; and Kempin et al., Nature 389:802-803, 1997). Alternatively, anendogenous tobacco nicotine demethylase gene may be replaced with a genethat does not have demethylase activity, for example, the sequence ofSEQ ID NO: 188.

Co-Suppression

A further desirable method of silencing gene expression isco-suppression (also referred to as sense suppression). This technique,which involves introduction of a nucleic acid, e.g., a nucleic acidfragment from a desired plant gene, such as any nucleic acid sequenceshown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, configured in thesense orientation, has been shown to effectively block the transcriptionof target genes (see, for example, Napoli et al., Plant Cell, 2:279-289,1990 and Jorgensen et al., U.S. Pat. No. 5,034,323).

Generally, sense suppression involves transcription of the introducedsequence. However, co-suppression may also occur where the introducedsequence contains no coding sequence per se, but only intron oruntranslated sequences or other such sequences substantially identicalto sequences present in the primary transcript of the endogenous gene tobe repressed. The introduced sequence generally will be substantiallyidentical to the endogenous gene targeted for repression. Such identityis typically greater than about 50%, but higher identities (for example,80% or even 95%) are preferred because they result in more effectiverepression. The effect of co-suppression may also be applied to otherproteins within a similar family of genes exhibiting homology orsubstantial homology. Segments from a gene from one plant can be useddirectly, for example, to inhibit expression of homologous genes indifferent plant species.

In sense suppression, the introduced sequence, requiring less thanabsolute identity, need not be full length, relative to either theprimary transcription product or to fully processed mRNA. A higherdegree of sequence identity in a shorter than full-length sequencecompensates for a longer sequence of lesser identity. Furthermore, theintroduced sequence need not have the same intron or exon pattern, andidentity of non-coding segments may be equally effective. Sequences ofat least 50 base pairs are preferred, with introduced sequences ofgreater length being more preferred (see, for example, those methodsdescribed by Jorgensen et al., U.S. Pat. No. 5,034,323).

Antisense Suppression

In antisense technology, a nucleic acid segment from the desired plantgene, such as any nucleic acid sequence shown in FIGS. 2 to 7 and SEQ IDNOS: 446 to 2193, is cloned and operably linked to an expression controlregion such that the antisense strand of RNA is synthesized. Theconstruct is then transformed into plants and the antisense strand ofRNA is produced. In plant cells, it has been shown that antisense RNAinhibits gene expression.

The nucleic acid segment to be introduced in antisense suppression isgenerally substantially identical to at least a portion of theendogenous gene or genes to be repressed, but need not be identical. Thenucleic acid sequences of the tobacco nicotine demethylase disclosedherein may be included in vectors designed such that the inhibitoryeffect applies to other proteins within a family of genes exhibitinghomology or substantial homology to the target gene. Segments from agene from one plant can be used, for example, directly to inhibitexpression of homologous genes in different tobacco varieties.

The introduced sequence also need not be full length relative to eitherthe primary transcription product or to fully processed mRNA. Generally,higher homology can be used to compensate for the use of a shortersequence. Moreover, the introduced sequence need not have the sameintron or exon pattern, and homology of non-coding segments will beequally effective. In general, such an antisense sequence will usuallybe at least 15 base pairs, preferably about 15-200 base pairs, and morepreferably 200-2,000 base pairs in length or greater. The antisensesequence may be complementary to all or a portion of the gene to besuppressed, and, as appreciated by those skilled in the art, theparticular site or sites to which the antisense sequence binds as wellas the length of the antisense sequence will vary, depending upon thedegree of inhibition desired and the uniqueness of the antisensesequence. A transcriptional construct expressing a plant negativeregulator antisense nucleotide sequence includes, in the direction oftranscription, a promoter, the sequence coding for the antisense RNA onthe sense strand, and a transcriptional termination region. Antisensesequences may be constructed and expressed as described, for example, invan der Krol et al. (Gene 72: 45-50, 1988); Rodermel et al. (Cell 55:673-681, 1988); Mol et al. (FEBS Lett. 268: 427-430, 1990); Weigel andNilsson (Nature 377: 5 495-500, 1995); Cheung et al., (Cell 82: 383-393,1995); and Shewmaker et al. (U.S. Pat. No. 5,107,065).

Dominant Negatives

Transgenic plants expressing a transgene encoding a dominant negativegene product of a tobacco gene product may be assayed in artificialenvironments or in the field to demonstrate that the transgene confersdownregulates a tobacco gene product in the transgenic plant. Dominantnegative transgenes are constructed according to methods known in theart. Typically, a dominant negative gene encodes a mutant negativeregulator polypeptide of a tobacco gene product which, whenoverexpressed, disrupts the activity of the wild-type enzyme.

Mutants

Plants having decreased expression or enzymatic activity of a tobaccogene product may also be generated using standard mutagenesismethodologies. Such mutagenesis methods include, without limitation,treatment of seeds with ethyl methylsulfate (Hildering and Verkerk, In,The use of induced mutations in plant breeding. Pergamon press, pp317-320, 1965) or UV-irradiation, X-rays, and fast neutron irradiation(see, for example, Verkerk, Neth. J. Agric. Sci. 19:197-203, 1971; andPoehlman, Breeding Field Crops, Van Nostrand Reinhold, New York(3.sup.rd ed), 1987), use of transposons (Fedoroff et al., 1984; U.S.Pat. No. 4,732,856 and U.S. Pat. No. 5,013,658), as well as T-DNAinsertion methodologies (Hoekema et al., 1983; U.S. Pat. No. 5,149,645).The types of mutations that may be present in a tobacco gene include,for example, point mutations, deletions, insertions, duplications, andinversions. Such mutations desirably are present in the coding region ofa tobacco gene; however mutations in the promoter region, and intron, oran untranslated region of a tobacco gene may also be desirable.

For instance, T-DNA insertional mutagenesis may be used to generateinsertional mutations in a tobacco gene to downregulate the expressionof the gene. Theoretically, about 100,000 independent T-DNA insertionsare required for a 95% probability of getting an insertion in any givengene (McKinnet, Plant J. 8:613-622, 1995; and Forsthoefel et al., Aust.J. Plant Physiol. 19:353-366, 1992). T-DNA tagged lines of plants may bescreened using polymerase chain reaction (PCR) analysis. For example, aprimer can be designed for one end of the T-DNA and another primer canbe designed for the gene of interest and both primers can be used in thePCR analysis. If no PCR product is obtained, then there is no insertionin the gene of interest. In contrast, if a PCR product is obtained, thenthere is an insertion in the gene of interest.

Expression of a mutated tobacco gene product may be evaluated accordingto standard procedures (for example, those described herein) and,optionally, may be compared to expression of the non-mutated enzyme.When compared to non-mutated plants, mutated plants having decreasedexpression of a gene encoding a tobacco gene product are desirableembodiments of the present invention. A plant having a mutation in anyof the nucleic acid sequences described herein may be used in a breedingprogram as described herein.

Overexpression of Constitutive or Ethylene or Senescence InducedSequences

Overexpression of a nucleic acid sequence of the invention (e.g., anucleic acid sequence shown in FIG. 1, FIGS. 3 to 7, FIGS. 10 to 158,FIGS. 162 to 170, FIGS. 172-1 to 172-19, and FIGS. 173-1 to 173-294, orfragments thereof) can be used to increase desirable traits in theNicotiana line or in a tobacco product made from a plant of that line.In particular, overexpression of the nucleic acid sequences of theinvention, and/or their translation products, may be used to increasethe biosynthesis of desirable flavor and aroma products that result fromsecondary metabolites. Further overexpression of a nucleic acid sequencethat encodes a tobacco polypeptide may be used to increase expression ofthe polypeptide within Nicotiana lines.

Additional desirable traits that may be conferred to a Nicotiana line byoverexpressing a nucleic acid sequence of the invention includeresistance to bacterial wilt, -GranvIDe wilt, Fusarium wilt, potatovirus Y, tobacco mosaic virus, tobacco etch virus, tobacco vein mottlingvirus, alfalfa mosaic viruses, wildfire, root-knot nematode, Southernroot knot nematode, cyst nematode, black root rot, blue mold, race 0black shank fungus, and race I, black shank fungus. Other desirabletraits that may be enhanced in a Nicotiana plant by overexpressing anucleic acid sequence of the invention include increased yield and/orgrade, better curability, harvestability, holding ability, leaf quality,or curing quality, increased or reduced height, altered time of maturity(e.g., early maturing, early to medium maturing, medium maturing, mediumto late maturing, or late maturing), increased or reduced stalk size,and an increase or reduction in the number of leaves per plant.

Plant Promoters

A desirable promoter is a caulimovirus promoter, for instance, acauliflower mosaic virus (CaMV) promoter or the cassaya vein mosaicvirus (CsVMV) promoter. These promoters confer high levels of expressionin most plant tissues, and the activity of these promoters is notdependent on virally encoded proteins. CaMV is a source for both the 35Sand 19S promoters. Examples of plant expression constructs using thesepromoters are known in the art. In most tissues of transgenic plants,the CaMV 35S promoter is a strong promoter. The CaMV promoter is alsohighly active in monocots. Moreover, activity of this promoter can befurther increased (i.e., between 2-10 fold) by duplication of the CaMV35S promoter.

Other useful plant promoters include, without limitation, the nopalinesynthase (NOS) promoter, the octopine synthase promoter, figwort mosiacvirus (FMV) promoter, the rice actin promoter, and the ubiquitinpromoter system.

Exemplary monocot promoters include, without limitation, commelinayellow mottle virus promoter, sugar cane badna virus promoter, ricetungro bacilliform virus promoter, maize streak virus element, and wheatdwarf virus promoter.

For certain applications, it may be desirable to produce a tobacco geneproduct, such as a dominant negative mutant gene product, in anappropriate tissue, at an appropriate level, or at an appropriatedevelopmental time; For this purpose, there are assortments of genepromoters, each with its own distinct characteristics embodied in itsregulatory sequences, shown to be regulated in response to induciblesignals such as the environment, hormones, and/or developmental cues.These include, without limitation, gene promoters that are responsiblefor heat-regulated gene expression, light-regulated gene expression (forexample, the pea rbcS-3A; the maize rbcS promoter; the chlorophyllalb-binding protein gene found in pea; or the Arabssu promoter),hormone-regulated gene expression (for example, the abscisic acid (ABA)responsive sequences from the Em gene of wheat; the ABA-inducible HVA1and HVA22, and rd29A promoters of barley and Arabidopsis; andwound-induced gene expression (for example, of wunl), organ specificgene expression (for example, of the tuber-specific storage proteingene; the 23-kDa zein gene from maize described by; or the French beanβ-phaseolin gene), or pathogen-inducible promoters (for example, thePR-l, prp-l, or β-1,3 glucanase promoters, the fungal-inducible wirlapromoter of wheat, and the nematode-inducible promoters, TobRB7-5A andHmg-l, of tobacco arid parsley, respectively).

Plant Expression Vectors

Typically, plant expression vectors include (1) a cloned plant geneunder the transcriptional control of 5′ and 3′ regulatory sequences and(2) a dominant selectable marker. Such plant expression vectors may alsocontain, if desired, a promoter regulatory region (for example, oneconferring inducible or constitutive, pathogen- or wound-induced,environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation, signal.

Plant expression vectors may also optionally include RNA processingsignals, e.g., introns, which have been shown to be important forefficient RNA synthesis and accumulation. The location of the RNA splicesequences can dramatically influence the level of transgene expressionin plants. In view of this fact, an intron may be positioned upstream ordownstream of a tobacco nicotine demethylase coding sequence in thetransgene to alter levels of gene expression.

In addition to the aforementioned 5′ regulatory control sequences, theexpression vectors may also include regulatory control regions which aregenerally present in the 3′ regions of plant genes. For example, the 3′terminator region may be included in the expression vector to increasestability of them RNA. One such terminator region may be derived fromthe PI-II terminator region of potato. In addition, other commonly usedterminators are derived from the octopine or nopaline synthase signals.

The plant expression vector also typically contains a dominantselectable marker gene used to identify those cells that have becometransformed. Useful selectable genes for plant systems include theaminoglycoside phosphotransferase gene of transposon Tn5 (Aph II), genesencoding antibiotic resistance genes, for example, those encodingresistance to hygromycin, kanamycin, bleomycin, neomycin, G418,streptomycin, or spectinomycin. Genes required for photosynthesis mayalso be used as selectable markers in photosynthetic-deficient strains.Finally, genes encoding herbicide resistance may be used as selectablemarkers; useful herbicide resistance genes include the bar gene encodingthe enzyme phosphinothricin acetyltransferase and conferring resistanceto the broad-spectrum herbicide Basta® (Bayer Cropscience DeutschlandGmbH, Langenfeld, Germany). Other selectable markers include genes thatprovide resistance to other such herbicides such as glyphosate and thelike, and imidazolinones, sulfonylureas, triazolopyrimidine herbicides,such as chlorosulfron, bromoxynil, dalapon, and the like. Furthermore,genes encoding dihydrofolate reductase may be used in combination withmolecules such as methatrexate.

Efficient use of selectable markers is facilitated by a determination ofthe susceptibility of a plant cell to a particular selectable agent anda determination of the concentration of this agent which effectivelykills most, if not all, of the transformed cells. Some usefulconcentrations of antibiotics for tobacco transformation include, forexample, 20-100 μg/ml (kanamycin), 20-50 μg/ml (hygromycin), or 5-10μg/ml (bleomycin). A useful strategy for selection of transformants forherbicide resistance is described, for example, by Vasil (Cell Cultureand Somatic Cell Genetics of Plants, Vol I, II, III LaboratoryProcedures and Their Applications, Academic Press, New York, 1984).

In addition to a selectable marker, it may be desirable to use areporter gene. In some instances a reporter gene may be used without aselectable marker. Reporter genes are genes which are typically notpresent or expressed in the recipient organism or tissue. The reportergene typically encodes for a protein which provide for some phenotypicchange or enzymatic property. Examples of such genes are provided inWeising et al. (Ann. Rev. Genetics 22:421, 25 1988), which isincorporated herein by reference. Preferred reporter genes includewithout limitation glucuronidase (GUS) gene and GFP genes.

Upon construction of the plant expression vector, several standardmethods are available for introduction of the vector into a plant host,thereby generating a transgenic plant. These methods include (1)Agrobacterium-mediated transformation (A. tumefaciens or A. rhizogenes)(see, for example, Lichtenstein and Fuller In: Genetic Engineering, vol6, PWJ Rigby, ed, London, Academic Press, 1987; and Lichtenstein, C. P.,and Draper, J., In: DNA Cloning, Vol II, D. M. Glover, ed, Oxford, IRIPress, 1985; U.S. Pat. Nos. 4,693,976, 4,762,785, 4,940,838, 5,004,863,5,104,310, 5,149,645, 5,159,135, 5,177,010, 5,231,019, 5,463,174,5,469,976, and 5,464,763; and European Patent Numbers 0131624, 0159418,0120516, 0176112, 0116718, 0290799, 0292435, 0320500, and 0627752, andEuropean Patent Application Numbers 0267159 and 0604622,), (2) theparticle delivery system (see, for example, U.S. Pat. Nos. 4,945,050 and5,141,131), (3) microinjection protocols, (4) polyethylene glycol (PEG)procedures, (5) liposome-mediated DNA uptake, (6) electroporationprotocols (see, for example, WO 87/06614 and U.S. Pat. Nos. 5,384,253,5,472,869, 5,641,664, 5,679,558, 5,712,135, 6,002,070, and 6,074,877,(7) the vortexing method, or (8) the so-called whiskers methodology(see, for example, Coffee et al., U.S. Pat. Nos. 5,302,523 and5,464,765). The type of plant tissue that may be transformed with anexpression vector includes embryonic tissue, callus tissue type I andII, hypocotyls, meristem, and the like.

Once introduced into the plant tissue, the expression of the structuralgene may be assayed by any means known to the art, and expression may bemeasured as mRNA transcribed, protein synthesized, or the amount of genesilencing that occurs as determined by metabolite monitoring viachemical analysis of secondary alkaloids in tobacco (as describedherein; see also U.S. Pat. No. 5,583,021 which is hereby incorporated byreference). Techniques are known for the in vitro culture of planttissue, and in a number of cases, for regeneration into whole plants(see, e.g., U.S. Pat. Nos. 5,595,733 and 5,766,900). Procedures fortransferring the introduced expression complex to commercially usefulcultivars are known to those skilled in the art.

Once plant cells expressing the desired level of a desirable geneproduct are obtained, plant tissues and whole plants can be regeneratedtherefrom using methods and techniques well-known in the art. Theregenerated plants are then reproduced by conventional means and theintroduced genes can be transferred to other strains and cultivars byconventional plant breeding techniques.

Transgenic tobacco plants may incorporate a nucleic acid of any portionof the genomic gene in different orientations for eitherdown-regulation, for example, antisense orientation, or over-expression,for example, sense orientation. Over-expression of the nucleic acidsequence that encodes the entire or a functional part of an amino acidsequence of a full-length tobacco gene is desirable for increasing theexpression of the gene product within Nicotiana lines.

Determination or Transcriptional or Translational Levels or a TobaccoGene

Gene expression may be measured, for example, by standard Northern blotanalysis (Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y., (2001), and. Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.,(1989) using a tobacco gene or gene fragment as a hybridization probe.Determination of RNA expression levels may also be aided by reversetranscription PCR (rtPCR), including quantitative rtPCR (see, e.g.,Kawasaki et al., in PCR Technology: Principles and Applications of DNAAmplification (H. A. Erlich, Ed.) Stockton Press (1989); Wang et al. inPCR Protocols: A Guide to Methods and Applications (M. A. Innis, et al.,Eds.) Academic Press (1990); and Freeman et al., Biotechniques 26:112-122 and 124-125, 1999). Additional well-known techniques fordetermining expression of a tobacco gene include in situ hybridization,and fluorescent in situ hybridization (see, e.g., Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., (2001). The above standard techniques are also useful to comparethe expression level between plants, for example, between a plant havinga mutation in a tobacco gene and a control plant.

If desired, expression of a tobacco gene (e.g., a nucleic acid sequenceshown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, or a fragmentthereof) may be measured at the level of protein production using thesame general approach and standard protein analysis techniques includingBradford assays, spectrophotometric assays, and immunological detectiontechniques, such as Western blotting or immunoprecipitation with anantibody specific for the desirable polypeptide (Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y.,(2001), and Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, N.Y., (1989).

The activity of any polypeptide described herein may be assayed usingstandard methods in the art. For example, the activity of a p450 istypically assayed using fluorescent-based assays (see, e.g., Donato etal. Drug Metab Dispos. 32:699-706, 2004). In particular, the activity ofa nicotine demethylase may be assayed as described herein using yeastmicrosome assays.

Identification of Modulators of a Tobacco Gene Product

Isolation of a cDNA also facilitates the identification of moleculesthat increase or decrease expression the gene product. According to oneapproach, candidate molecules are added at varying concentrations to aculture medium of cells (for example, prokaryotic cells such as E. colior eukaryotic cells such as yeast, mammalian, insect, or plant cells)expressing a tobacco mRNA. Gene product expression is then measured inthe presence and absence of a candidate molecule using standard methodssuch as those set forth herein.

Candidate modulators may be purified. (or substantially purified)molecules or may be one component of a mixture of compounds. In a mixedcompound assay, gene product expression is tested against progressivelysmaller subsets of the candidate compound pool (for example, produced bystandard purification techniques, for example, HPLC) until a singlecompound or minimal compound mixture is demonstrated to alter tobacconicotine demethylase gene expression. In one embodiment of theinvention, a molecule that promotes a decrease gene product expressionis considered particularly desirable. Modulators found to be effectiveat the level of gene product expression or activity may be confirmed asuseful in planta.

For agricultural uses, the molecules, compounds, or agents identifiedusing the methods disclosed herein may be used as chemicals applied assprays or dusts on the foliage of plants. The molecules, compounds, oragents may also be applied to plants in combination with anothermolecule which affords some benefit to the plant.

Uses

Regulation of the endogenous gene corresponding to any of the sequencesdescribed herein by, for example, gene silencing may result in morevaluable plants or plant products. In particular, sequences identifiedherein as ethylene-induced or senescence-related (e.g., those having thesequence of SEQ ID NOS: 4, 40, 44, 52, 54, 60, 70, 104, 138, 140, 158,162, 188, 212, 226, 234, and 288 or a nucleic acid sequence shown inFIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, or a fragment thereof) may beused to affect metabolic pathways involved in the formation of numeroussecondary metabolites including terpenoids, polyphenols, alkaloids, etc.that affect end-product quality traits. Similarly the genes identifiedherein may be used to regulate metabolic pathways associated with therate and type of dry matter accumulated during senescence or thepartitioning of dry matter within the plant during senescence.Regulating the genes identified herein may also be used to affectmetabolic pathways involved in determining senescence rates, theuniformity of senescence within a leaf and among leaves of a singleplant, and the induction of senescence by agents or activities thatstimulate or activate the genes identified herein, and, thereby, controlthe quality of a product or article of manufacture that includes a leafor other plant component.

The promoter region of a gene described herein may be used to driveexpression of any desirable gene product to improve crop quality orenhance specific traits. A promoter that is inducible and expressedduring a particular period of the plant's life cycle can be used inconstructs for introduction into the plant to express unique genesinvolved in the biosynthesis of flavor and aroma products that resultfrom secondary metabolites. A tobacco gene promoter may also be used toincrease or modify the expression of structural carbohydrates orproteins that affect end-use properties. Further, a tobacco genepromoter could be combined with heterologous genes that include genesinvolved in the biosynthesis of nutritional products, pharmaceuticalagents, or industrial materials. Regulation of a promoter sequence mayalso be used to downregulate endogenous tobacco genes, including genesinvolved in alkaloid biosynthesis and/or in other pathways. Desirably, atobacco gene promoter region or other transcriptional regulatory regionis used to alter chemical properties such as nornicotine content andnitrosamine levels in a plant. In addition, promoter motifs, which canreadily be identified in a promoter sequence using standard methods inthe art, may be used to identify factors that associate with or regulatethe expression of a tobacco gene product, e.g., a p450. Moreover, any ofthe sequences of the present invention (e.g., the nucleic acid sequencesshown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193; or fragments thereof)may be used in methods that reduce gene expression or alter enzymaticactivity of a gene product, such as a p450, using standard techniquesdescribed herein. Such techniques include, without limitation, RNAinterference, triple strand interference, ribozymes, homologousrecombination, virus-induced gene silencing, antisense andco-suppression technologies, expression of a dominant negative geneproduct, and the generation of mutated genes using standard mutagenesistechniques. For example, reducing p450 expression or altering p450enzymatic activity may be used to alter fatty acids that are involved inplant-pathogen interactions and disease resistance or may be used toalter a plant's profile of selected fatty acids and thereby alter theflavor or aroma of the plant or plant component.

Furthermore, using standard methods, any portion of a tobacco gene,including the promoter, the coding sequence, an intron, or a 3′UTR, or afragment thereof, can be used as a genetic marker to isolate relatedgenes, promoters or regulatory regions, for screening for the relatedgene in other tobacco or Nicotiana species, or for determining whether aplant has a mutation in a corresponding endogenous gene. A portion of atobacco gene may also be used to monitor gene flow through a breedingeffort to track intergression or loss of a particular gene.

For example, Nicotiana tabacum is an allotetraploid, as are several ofthe other Nicotiana species, and the genetic markers could be used toidentify homologous genes or related genes in the parental genomedifferent from the genome in which the original gene resides. A markerfor the related gene could be also be used to screen existing tobaccogermplasm, segregating or synthetic populations created byhybridizations, populations created from mutagenic treatments or fromvarious tissue culture methods. As such, the nucleic acid sequencesdescribed herein (e.g., the nucleic acid sequences shown in FIGS. 2 to 7and SEQ ID NOS: 446 to 2193, or fragments thereof) may be used toidentify or affect genes involved in disease or insect resistance,flavor and aroma properties, herbicide tolerance, quality factorsrelated to undesirable constituents, or that increase leaf yield, oraffect leaf or plant components, such as lignins, cellulose, etc.,related to structural traits or fiber content.

Products

Tobacco products having a reduced amount of nitrosamine content aremanufactured using any of the tobacco plant material described hereinaccording to standard methods known in the art. In one embodiment,tobacco products are manufactured using tobacco plant material obtainedfrom a cured tobacco plant. The cured tobacco plant may contain or havebeen bred to contain reduced nicotine demethylase activity. For example,the cured tobacco plant may be a tobacco plant resulting from a crossincluding a tobacco plant identified as having variant expression ofnicotine demethylase. Desirably the tobacco product has a reduced amountof nornicotine or NNN of less than about 5 mg/g, 4.5 mg/g, 4.0 mg/g, 3.5mg/g, 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 μg/g, 500μg/g, 250 μg/g, 100 μg/g, 75 μg/g, 50 μg/g, 25 μg/g, 10 μg/g, 7.0 μg/g,5.0 μg/g, 4.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, 0.4 μg/g, 0.2 μg/g,0.1 μg/g, 0.05 μg/g, or 0.01 μg/g or wherein the percentage of secondaryalkaloids relative to total alkaloid content contained therein is lessthan 90%, 70%, 50%, 30%, 10%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.75%, 0.5%,0.25%, or 0.1%. The phrase “a reduced amount” refers to an amount ofnornicotine or NNN or both in a tobacco plant or plant component or atobacco product that is less than what would be found in a wild-typetobacco plant or plant component or tobacco product from the samevariety of tobacco, processed in the same manner, which was not madetransgenic for reduced nornicotine or NNN. In one example, a wild-typetobacco plant of the same variety that has been processed in the samemanner is used as a control to measure whether a reduction ofnornicotine or NNN or both has been obtained by the methods describedherein. In another example, plants having a reduced amount ofnitrosamine content are evaluated using standard methods, for instance,by monitoring the presence or absence of a gene or gene product, e.g., anicotine demethylase; or a particular mutation in a gene. In stillanother example, nitrosamine content of plants resulting from a breedingprogram are compared to the nitrosamine content of the recipient line ordonor line, or both, used to breed the plant having the reduced amountof nitrosamine. Other suitable controls known in the art are also usedas needed. Levels of nornicotine and NNN or both are measured accordingto methods well known in the tobacco art.

The following examples illustrate methods for carrying out the inventionand should be understood to be illustrative of, but not limiting upon,the scope of the invention which is defined in the appended claims.

Example 1 Development of Plant Tissue and Ethylene Treatment PlantGrowth

Plants were seeded in pots and grown in a greenhouse for 4 weeks. The4-week old.seedlings were transplanted into individual pots and grown inthe greenhouse for 2 months. The plants were watered 2 times a day withwater containing 150 ppm NPK fertilizer during growth. The expandedgreen leaves were detached from plants to do the ethylene treatmentdescribed below.

Cell Line 78379

Tobacco line 78379, which is a Burley tobacco line released by theUniversity of Kentucky was used as a source of plant material. Onehundred plants were cultured as standard in the art of growing tobacco,transplanted, and tagged with a distinctive number (1-100).Fertilization and field management were conducted as recommended.

Three quarters of the 100 plants converted between 20 and 100% of thenicotine to nornicotine. One quarter of the 100 plants converted lessthan 5% of the nicotine to nornicotine. Plant number 87 had the leastconversion (2%) while plant number 21. had 100% conversion. Plantsconverting less than 3% were classified as non-converters.Self-pollinated seed of plant number 87 and plant number 21, as well ascrossed (21×87 and 87×21) seeds were made to study genetic andphenotypic differences. Plants from selfed 21 were converters, and 99%of selfs from 87 were non-converters. The other 1% of the plants from 87showed low conversion (5-15%). Plants from reciprocal crosses were allconverters.

Cell Line 4407

Nicotiana line 4407, which is a Burley line, was used as a source ofplant material. Uniform and representative plants (100) were selectedand tagged. Of the 100 plants 97 were non-converters and three wereconverters. Plant number 56 had the least amount of conversion (1.2%)and plant number 58 had the highest level of conversion (96%).Self-pollinated seeds and crossed seeds were made with these two plants.

Plants from selfed-58 segregated with 3:1 converter to non-converterratio. Plants 58-33 and 58-25 were identified as homozygous converterand nonconverter plant lines, respectively. The stable conversion of58-33 was confirmed by analysis of its progeny.

Cell Line PBLB01

PBLB01 is a Burley line developed by ProfiGen, Inc. and was used as asource of plant material. The converter plant was selected fromfoundation seeds of PBLB01.

Ethylene Treatment Procedures

Green leaves were detached from 2-3 month greenhouse grown plants andsprayed with 0.3% ethylene solution (Prep brand Ethephon(Rhone-Poulenc>>. Each sprayed leaf was hung in a curing rack equippedwith humidifier and covered with plastic. During the treatment, thesample leaves were periodically sprayed with the ethylene solution.Approximately 24-48 hour post ethylene treatment, leaves were collectedfor RNA extraction. Another sub-sample was taken for metabolicconstituent analysis to determine the concentration of leaf metabolitesand more specific constituents of interest such as a variety ofalkaloids.

As an example, alkaloids analysis could be performed as follows. Samples(0.1 g) were shaken at 150 rpm with 0.5 ml 2N NaOH, and a 5 mlextraction solution which contained quinoline as an internal standardand methyl t-butyl ether. Samples were analyzed on a HP 6890 GC equippedwith a FID detector. A temperature of 250° C. was used for the detectorand injector. An HP column (30m-0.32 nm-1 mm) consisting of fused silicacrosslinked with 5% phenol and 95% methyl silicon was used at atemperature gradient of 110-185° C. at 10° C. per minute. The column wasoperated at 100° C. with a flow rate of 1.7 cm³ min⁻¹ with a split ratioof 40:1 with a 2:1 injection volume using helium as the carrier gas.

Example 2 RNA Isolation

For RNA extractions, middle leaves from two-month old greenhouse grownplants were treated with ethylene as described above. The 0 and 24-48hours samples were used for RNA extraction. In some cases, leaf samplesunder the senescence process were taken from the plants 10 days postflower-head removal. These samples were also used for extraction. TotalRNA was isolated using Rneasy Plant Mini Kit® (Qiagen, Inc., Valencia,Calif.) according to the manufacturer's protocol.

The tissue sample was ground under liquid nitrogen to a fine powderusing a DEPC treated mortar and pestle. Approximately 100 milligrams ofground tissue were transferred to a sterile 1.5 ml Eppendorf tube. Thissample tube was placed in liquid nitrogen until all samples werecollected. Then, 450 μl of Buffer RLT as provided in the kit (with theaddition of Mercaptoethanol) was added to each individual tube. Thesample was vortexed vigorously and incubated at 56° C. for 3 minutes.The lysate was then applied to the QIAshredder® spin column sitting in a2 ml collection tube, and centrifuged for 2 minutes at maximum speed.The flow through was collected and 0.5 volume of ethanol was added tothe cleared lysate. The sample was mixed well and transferred to anRneasy® mini spin column sitting in a 2 μl collection tube. The samplewas centrifuged for 1 minute at 10,000 rpm. Next, 700 μl of buffer RW1was pipetted onto the Rneasy® column and centrifuged for 1 minute at10,000 rpm. Buffer RPE was pipetted onto the Rneasy® column in a newcollection tube and centrifuged for 1 minute at 10,000 rpm. Buffer RPEwas again, added to the Rneasy® spin column and centrifuged for 2minutes at maximum speed to dry the membrane. To eliminate any ethanolcarry over, the membrane was placed in a separate collection tube andcentrifuged for an additional 1 minute at maximum speed. The Rneasy®column was transferred into a new 1.5 ml collection tube, and 40 μl ofRnase-free water was pipetted directly onto the Rneasy® membrane. Thisfinal elute tube was centrifuged for 1 minute at 10,000 rpm. Quality andquantity of total RNA was analyzed by denatured formaldehyde gel andspectrophotometer.

Poly(A)RNA was isolated using Oligotex® poly A+ RNA purification kit(Qiagen Inc.) following the manufacturer's protocol. About 200 μg totalRNA in 250 μl maximum volume was used. A volume of 250 μl of Buffer OBBand 15 μl of Oligotex® suspension was added to the 250 μl of total RNA.The contents were mixed thoroughly by pipetting and incubated for 3minutes at 70° C. on a heating block. The sample was then placed at roomtemperature for approximately 20 minutes. The Oligotex®: mRNA complexwas pelleted by centrifugation for 2 minutes at maximum speed. All but50 μl of the supernatant was removed from the microcentrifuge tube. Thesample was treated further by OBB buffer. The Oligotex®: mRNA pellet wasresuspended in 400 μl of Buffer OW2 by vortexing. This mix wastransferred onto a small spin column placed in a new tube andcentrifuged for 1 minute at maximum speed. The spin column wastransferred to a new tube and an additional 4000 μL of Buffer OW2 wasadded to the column. The tube was then centrifuged for 1 minute atmaximum speed. The spin column was transferred to a final 1.5 mlmicrocentrifuge tube. The sample was eluted with 60 μl of hot (70° C.)Buffer OEB. Poly A product was analyzed by denatured formaldehyde gelsand spectrophotometric analysis.

Example 3 Reverse-Transcription-PCR

First strand cDNA was produced using SuperScript reverse transcriptasefollowing the manufacturer's protocol (Invitrogen, Carlsbad, Calif.).The poly A+ enriched RNA/oligo dT primer mix consisted of less than 5 μgof total RNA, 1 μl of 10 mM dNTP mix, 1 μl of Oligo d(T)₁₂₋₁₈ (0.5μg/μl), and up to 10 μl of DEPC-treated water. Each sample was incubatedat 65° C. for 5 minutes, then placed on ice for at least 1 minute. Areaction mixture was prepared by adding each of the following componentsin order: 2 μl 10×RT buffer, 4 μl of 25 mM MgCl₂, 2 μl of 0.1 M DTT, and1 μl of RNase OUT Recombinant RNase Inhibitor. An addition of 9 μl ofreaction mixture was pipetted to each RNA/primer mixn.lfe and gentlymixed. It was incubated at 42° C. for 2 minutes and 1 μl of Super ScriptII RT was added to each tube. The tube was incubated for 50 minutes at42° C. The reaction was terminated at 70° C. for 15 minutes and chilledon ice. The sample was collected by centrifugation and 1 μl of RNase Hwas added to each tube and incubated for 20 minutes at 37° C. The secondPCR was carried out with 200 pmoles of forward primer and 100 pmolesreverse primer (mix of 18 nt oligo d(T) followed by 1 random base).

Reaction conditions were 94° C. for 2 minutes and then 40 cycles of PCRat 94° C. for 1 minute, 45° C. to 60° C. for 2 minutes, 72° C. for 3minutes, with a 72° C. extension for an extra 10 min. Ten microliters ofthe amplified sample were analyzed by electrophoresis using a 1% agarosegel. The correct size fragments were purified from agarose gel.

Example 4 Generation of PCR Fragment Populations

PCR fragments from Example 3 were ligated into a pGEM-T Easy Vector(Promega, Madison, Wis.) following the manufacturer's instructions. Theligated product was transformed into JM109 competent cells and plated onLB media plates for blue/white selection. Colonies were selected andgrown in a 96 well plate with 1.2 ml of LB media overnight at 37° C.Frozen stock was generated for all selected colonies. Plasmid DNA waspurified from plates using Beckman's Biomeck 2000 miniprep robotics withWizard SV Miniprep kit (Promega). Plasmid DNA was eluted with 100 μlwater and stored in a 96 well plate. Plasmids were digested by EcoRI andwere analyzed using 1% agarose gel to confirm the DNA quantity and sizeof inserts. Plasmids containing a 400-600 by insert were sequenced usinga CEQ 2000 sequencer (Beckman, Fullerton, Calif.). The sequences werealigned with GenBank database by BLAST search (see, e.g., FIGS. 159A to159K). The p450 related fragments were identified and further analyzed.Alternatively, p450 fragments were isolated from subtraction libraries.These fragments were also analyzed as described above.

Example 5 cDNA Library Construction

A cDNA library was constructed by preparing total RNA from ethylenetreated leaves as follows. First, total RNA was extracted from ethylenetreated leaves of tobacco line 58-33 using a modified acid phenol andchloroform extraction protocol. The protocol was modified to use onegram of tissue that was ground and subsequently vortexed in 5 ml ofextraction buffer (100 mM Tris-HCl, pH 8.5; 200 mM NaCl; 10 mM EDTA;0.5% SDS) to which 5 ml phenol (pH 5.5) and 5 ml chloroform was added.The extracted sample was centrifuged and the supernatant was saved. Thisextraction step was repeated 2-3 times until the supernatant appearedclear. Approximately 5 ml of chloroform was added to remove traceamounts of phenol. RNA was precipitated from the combined supernatantfractions by adding a 3-fold volume of ethanol and 1110 volume of 3MNaOAc (pH 5.2) and storing at −20° C. for 1 hour. After transfer to aCorex glass container the RNA fraction was centrifuged at 9,000 RPM for45 minutes at 4° C. The pellet was washed with 70% ethanol and spun for5 minutes at 9,000 RPM at 4° C. After drying the pellet, the pelletedRNA was dissolved in 0.5 ml RNase free water. The quality and quantityof total RNA was analyzed by denatured formaldehyde gel andspectrophotometer, respectively.

The resultant total RNA was used to isolate poly A+ RNA using anOligo(dT) cellulose protocol (Invitrogen) and microcentrifuge spincolumns (Invitrogen) by the following protocol. Approximately twenty mgof total RNA was twice subjected to purification to obtain high qualitypoly A+ RNA. Poly A+ RNA product was analyzed by performing denaturedformaldehyde gel and subsequent RT-PCR of known full-length genes toensure high quality of mRNA.

Next, poly A+ RNA was used as template to produce a cDNA libraryemploying cDNA synthesis kit, ZAP-cDNA synthesis kit, and ZAP-cDNAGigapack ID gold cloning kit (Stratagene, La Jolla, Calif.). The methodinvolved following the manufacturer's protocol as specified.Approximately 8 μg of poly A+ RNA was used to construct cDNA library.Analysis of the primary library revealed about 2.5×106-1×107 pfu. Aquality background test of the library was completed by complementationassays using IPTG and X-gal, where recombinant plaques was expressed atmore than 100-fold above the background reaction.

A more quantitative analysis of the library by random PCR showed thataverage size of insert cDNA was approximately 1.2 kb. The method used atwo-step PCR method. For the first step, reverse primers were designedbased on the preliminary sequence information obtained from p450fragments. The designed reverse primers and T3 (forward) primers wereused to amplify corresponding genes from the cDNA library PCR reactionswere subjected to agarose electrophoresis and the corresponding bands ofhigh molecular weight were excised, purified, cloned and sequenced. Inthe second step, new primers designed from 5′UTR or the start codingregion of p450 as the forward primers together with the reverse primers(designed from 3′UTR of p450) were used in the subsequent PCR to obtainfull-length p450 clones.

The p450 fragments were generated by PCR amplification from theconstructed cDNA library as described in Example 3 with the exception ofthe reverse primer. The T7 primer located on the plasmid downstream ofcDNA inserts was used as a reverse primer. PCR fragments were isolated,cloned and sequenced as described in Example 4.

Full-length p450 genes were isolated by this PCR method from constructedcDNA library. Gene specific reverse primers (designed from thedownstream sequence of p450 fragments) and a forward primer (T3 onlibrary plasmid) were used to clone the full-length genes. PCR fragmentswere isolated, cloned and sequenced. If necessary, a second PCR step wasapplied. In the second step, new forward primers designed from 5′UTR ofcloned p450s together with the reverse primers designed from 3′UTR ofp450 clones were used in the subsequent PCR reactions to obtainfull-length p450 clones. The clones were subsequently sequenced.

Example 6 Characterization of Cloned Fragments Reverse Southern BlottingAnalysis

Nonradioactive large-scale reverse Southern blotting assays wereperformed on all p450 clones identified in above examples to detect thedifferential expression. It was observed that the level of expressionamong different p450 clusters was very different. Further real timedetection was conducted on those with high expression.

Nonradioactive Southern blotting procedures were conducted as follows.

1) Total RNA was extracted from ethylene treated and nontreatedconverter (58-33) and nonconverter (58-25) leaves using the QiagenRnaeasy kit as described in Example 2.

2) A probe was produced by biotin-tail labeling a single strand cDNAderived from poly A+ enriched RNA generated in above step. This labeledsingle strand cDNA was generated by RT-PCR of the converter andnonconverter total RNA (Invitrogen) as described in Example 3 with theexception of using biotinylated oligo dT as a primer (Promega). Thesewere used as a probe to hybridize with cloned DNA.

3) Plasmid DNA was digested with restriction enzyme EcoRI and run onagarose gels. Gels were simultaneously dried and transferred to twonylon membranes (Biodyne B). One membrane was hybridized with converterprobe and the other with nonconverter probe. Membranes wereUV-crosslinked (auto crosslink setting, 254 nm, Stratagene,Stratalinker) before hybridization.

Alternatively, the inserts were PCR amplified from each plasmid usingthe sequences located on both arms of p-GEM plasmid, T3 and SP6, asprimers. The PCR products were analyzed by running on a 96 wellReady-to-run agarose gels. The confirmed inserts were dotted on twonylon membranes. One membrane was hybridized with converter probe andthe other with nonconverter probe.

4) The membranes were hybridized and washed following the manufacturer'sinstructions with the modification of washing stringency (Enzo MaxSencekit, Enzo Diagnostics, Inc, Farmingdale, N.Y.). The membranes wereprehybridized with hybridization buffer (2×SSC buffered formamide,containing detergent and hybridization enhancers) at 42° C. for 30 minand hybridized with 10 μl denatured probe overnight at 42° C. Themembranes then were washed in IX hybridization wash buffer 1 time atroom temperature for 10 min and 4 times at 68° C. for 15 min. Themembranes were ready for the detection procedure.

5) The washed membranes were detected by alkaline phosphatase labelingfollowed by NBTIBCIP colometric detection as described in manufacturer'sdetection procedure (Enzo Diagnostics, Inc.). The membranes were blockedfor one hour at room temperature with Ix blocking solution, washed 3times with IX detection reagents for 10 min, washed 2 times with Ixpredevelopment reaction buffer for 5 min and then developed the blots indeveloping solution for 30-45 min until the dots appear. All reagentswere provided by the manufacturer (Enzo Diagnostics, Inc). In addition,large-scale reverse Southern assay was also performed using KPL Southernhybridization and detection kit following the manufacturer'sinstructions (KPL, Gaithersburg, Md.).

Example 7 Characterization of Clones—Northern Blot Analysis

As an alternative to Southern blot analysis, some membranes werehybridized and detected as described in the example of Northern blottingassays. Northern hybridization was used to detect mRNA differentiallyexpressed in Nicotiana as follows.

A random priming method was used to prepare probes from cloned p450(Megaprime DNA Labelling Systems, Amersham Biosciences). The followingcomponents were mixed: 25 ng denatured DNA template; 4 ul of eachunlabeled dTTP, dGTP and dCTP; 5 uI of reaction buffer; p32-labelleddATP and 2 ul of Klenow I; and H₂O, to bring the reaction to 50 μl. Themixture was incubated in 37° C. for 1-4 hours, and stopped with 2 μl of0.5 M EDTA. The probe was denatured by incubation at 95° C. for 5minutes before use.

RNA samples were prepared from ethylene treated and non-treated freshleaves of several pairs of tobacco lines. In some cases poly A+ enrichedRNA was used. Approximately 15 μg total RNA or 1.8 μg mRNA (methods ofRNA and mRNA extraction as described in Example 5) were brought to equalvolume with DEPC H₂O (5-10 μl). The same volume ofloading buffer(1×MOPS; 18.5% Formaldehyde; 50% Formamide; 4% Ficoll400;Bromophenolblue) and 0.5 μl EtBr (0.5 μg/μl) were added. The sampleswere subsequently denatured in preparation for separation of the RNA byelectrophoresis.

Samples were subjected to electrophoresis on a formaldehyde gel (1%Agarose, 1×MOPS, 0.6 M Formaldehyde) with 1×MOP buffer (0.4 MMorpholinopropanesulfonic acid; 0.1 M Na-acetate-3×H20; 10 mM EDTA;adjust to pH 7.2 with NaOH). RNA was transferred to a Hybond-N+membrane(Nylon, Amersham Pharmacia Biotech) by capillary method in 10×SSC buffer(1.5 M NaCl; 0.15 M Na-citrate) for 24 hours. Membranes with RNA sampleswere UV-crosslinked (auto crosslink setting, 254 nm, Stratagene,Stratalinker) before hybridization.

The membrane was prehybridized for 1-4 hours at 42° C. with 5-10 mlprehybridization 10 buffer (5×SSC; 50% Formamide; 5×Denhardt's-solution;1% SDS; 100/μg/ml heat-denatured sheared non-homologous DNA). Oldprehybridization buffer was discarded, and new prehybridization bufferand probe were added. The hybridization was carried out overnight at 42°C. The membrane was washed for 15 minutes with 2×SSC at roomtemperature, followed by a wash with 2×SSC.

As illustrated in Table 1 below, Northern blots and reverse SouthernBlot were useful in determining which genes were induced by ethylenetreatment relative to non-induced plants. Interestingly, not allfragments were affected similarly in the converter and nonconverter.Some of the cytochrome p450 fragments were partially sequenced todetermine their structural relatedness. This information was used tosubsequently isolate and characterize full-length gene clones ofinterest.

TABLE I The Effect of Ethylene Treatment on mRNA Induction Induced mRNAExpression Ethylene Treatment Fragments Converter D56-AC7 (SEQ ID No:44) + D56-AG11 (SEQ ID No: 40) + D56-AC12 (SEQ ID No: 54) + D70A-AB5(SEQ ID No: 104) + D73-AC9 (SEQ ID No: 52) + D70A-AA12 (SEQ ID No:140) + D73A-AG3 (SEQ ID No: 138) + D34-52 (SEQ ID No: 70) + D56-AG6 (SEQID No: 60) +

Northern analysis was performed using full-length clones on tobaccotissue obtained from converter and nonconverter Burley lines that wereinduced by ethylene treatment. This analysis was used to identifyfull-length clones that showed elevated expression in ethylene inducedconverter lines relative to ethylene induced converter lines relative toethylene induced nonconverter Burley lines. By so doing, thefunctionality relationship of full-length clones may be determined bycomparing biochemical differences in leaf constituents between converterand non-converter lines.

As shown in Table 2 below, six clones showed significantly higherexpression, as denoted by ++ and +++, in converter ethylene treatedtissue in comparison to non-converter treated tissue, denoted by +. Allof these clones showed little or no expression in converter andnon-converter lines that were not ethylene treated.

TABLE 2 Clones with Elevated Expression in Converter Ethylene-TreatedTissue Full Length Clones Converter Nonconverter D101 BA2 (SEQ ID NO:288) ++ + D207 AA5 (SEQ ID NO: 212) ++ + D208 AC8 (SEQ ID NO: 226) +++ +D237 ADI (SEQ ID NO: 234) ++ + D89 ABI (SEQ ID NO: 158) ++ + D90A BB3(SEQ ID NO: 162) ++ +

Example 8 Immunodetection of Polypeptides Encoded by the Cloned Genes

Peptide regions corresponding to 20-22 amino acids in length from threep450 clones were selected for (1) having lower or no homology to otherclones and (2) having good hydrophilicity and antigenicity. The aminoacid sequences of the peptide regions selected from the respective p450clones are listed below. The synthesized peptides were conjugated withKHL (keyhole limpet hemocyanin) and then injected into rabbits. Antiserawere collected 2 and 4 weeks after the 4th injection (Alpha DiagnosticIntl. Inc. San Antonio, Tex.).

D234-AD1 DIDGSKSKLVKAHRKIDEILG (SEQ ID NO: 2266) D90a-BB3RDAFREKETFDENDVEELNY (SEQ ID NO: 163) D89-AB1 FKNNGDEDRHFSQKLGDLADKY(SEQ ID NO: 2267)

Antisera were examined for crossreactivity to target proteins fromtobacco plant tissue by Western Blot analysis. Crude protein extractswere obtained from ethylene treated (0 to 40 hours) middle leaves ofconverter and nonconverter lines. Protein concentrations of the extractswere determined using RC DC Protein Assay Kit (BIO-RAD) following themanufacturer's protocol.

Two micrograms of protein were loaded onto each lane and the proteinswere separated on 10%-20% gradient gels using the Laemmli SDS-PAGEsystem. The proteins were transferred from gels to PROTRANNitrocellulose Transfer Membranes (Schleicher & Schuell) with theTrans-Blot Semi-Dry cell (BIO-RAD). Target p450 proteins were detectedand visualized with the ECL Advance Western Blotting Detection Kit(Amersham Biosciences). Primary antibodies against the synthetic-KLHconjugates were made in rabbits. Secondary antibody against rabbit IgG,coupled with peroxidase, was purchased from Sigma. Both primary andsecondary antibodies were used at 1:1000 dilutions. Antibodies showedstrong reactivity to a single band on the Western Blots indicating thatthe antisera were monospecific to the target peptide of interest.Antisera were also crossreactive with synthetic peptides conjugated toKLH.

Example 9 Nucleic Acid Identity, Structure Relatedness of IsolatedNucleic Acid Fragments, and GeneChip® Hybridization

Over 100 cloned p450 fragments were sequenced in conjunction withNorthern blot analysis to determine their structural relatedness. Theapproach used forward primers based either of two common p450 motifslocated near the carboxyl-terminus of the p450 genes. The forwardprimers corresponded to cytochrome p450 motifs FXPERF (SEQ ID NO: 2268)or GRRXCP(A/G) (SEQ ID NO: 2269). The reverse primers used standardprimers from either the plasmid, SP6 or T7 located on both arms of Pgemplasmid, or a poly A tail. The protocol used is described below.

Spectrophotometry was used to estimate the concentration of startingdouble stranded DNA following the manufacturer's protocol (BeckmanCoulter). The template was diluted with water to the appropriateconcentration, denatured by heating at 95° C. for 2 minutes, andsubsequently placed on ice. The sequencing reaction was prepared on iceusing 0.5 to 10 μl of denatured DNA template, 2 μl of 1.6 pmole of theforward primer, 8 μl of DTCS Quick Start Master Mix and the total volumebrought to 20 μl with water. The thermocycling program consisted of 30cycles of the follow cycle: 96° C. for 20 seconds, 50° C. for 20seconds, and 60° C. for 4 minutes followed by holding at 4° C.

The sequencing reaction was stopped by adding 5 μl of stop buffer (equalvolume of 3M NaOAc and 100 mM EDTA and 1 μl of 20 mg/ml glycogen). Thesample was precipitated with 60 μl of cold 95% ethanol and centrifugedat 6000×g for 6 minutes. Ethanol was discarded. The pellet was washedtwice with 200 μl of cold 70% ethanol. After the pellet was dry, 40 μlof SLS solution were added and the pellet was resuspended. A layer ofmineral oil was overlaid and the sample was placed on the CEQ 8000Automated Sequencer for further analysis.

To verify nucleic acid sequences, the nucleic acid sequence wasre-sequenced in both directions using forward primers to the FXPERF (SEQID NO: 2268) or GRRXCP(A/G) (SEQ ID NO: 2269) region of the p450 gene orreverse primers to either the plasmid or poly A tail. All sequencing wasperformed at least twice in both directions.

The nucleic acid sequences of cytochrome p450 fragments were compared toeach other from the coding region corresponding to the first nucleicacid after the region encoding the GRRXCP(NG) (SEQ ID NO: 2269) motifthrough to the stop codon. This region was selected as an indicator ofgenetic diversity among p450 proteins. A large number of geneticallydistinct p450 genes, in excess of 70 genes, were observed, similar tothat of other plant species. Upon comparison of nucleic acid sequences,it was found that the genes could be placed into distinct sequencesgroups based on their sequence identity. It was found that the bestunique grouping of p450 members was determined to be those sequenceswith 75% nucleic acid identity or greater. (See e.g., Table 1 of the US2004/0162420 patent application publication, which is incorporatedherein by reference.) Reducing the percentage identity resulted insignificantly larger groups. A preferred grouping was observed for thosesequences with 81% nucleic acid identity or greater, a more preferredgrouping 91% nucleic acid identity or greater, and a most preferredgrouping for those sequences 99% nucleic acid identity of greater. Mostof the groups contained at least two members and frequently three ormore members. Others were not repeatedly discovered suggesting thatapproach taken was able to isolate both low and high expressing mRNA inthe tissue used.

Based on 75% nucleic acid identity or greater, two cytochrome p450groups were found to contain nucleic acid sequence identity topreviously tobacco cytochrome genes that are genetically distinct fromthose within the group. Group 23, showed nucleic acid identity, withinthe parameters used for Table 3A, to GenBank sequences GI: 1171579 (SEQID NO: 2270) (CAA64635) and GI:14423327 (SEQ ID NO: 2271) (orAAK62346).GI:1171579 (SEQ ID NO: 2270) had nucleic acid identity toGroup 23 members ranging 96.9% to 99.5% identity to members of Group 23while GI: 14423327 (SEQ ID NO: 2271) ranged 95.4% to 96.9% identity tothis group. The members of Group 31 had nucleic acid identity rangingfrom 76.7% to 97.8% identity to the GenBank reported sequence of GI:14423319 (SEQ ID NO: 2272) (AAK62342). None of the other p450 identitygroups of Table 3A contained parameter identity, as used in Table 3A, topreviously reported Nicotiana p450s genes.

A consensus sequence with appropriate nucleic acid degenerate probescould be derived for a group to preferentially identify and isolateadditional members of each group from Nicotiana plants.

TABLE 3A Nicotiana p450 Nucleic Acid Sequence Identity Groups GROUPFRAGMENTS 1 D58-BG7 (SEQ ID NO: 10), D58-AB1 (SEQ ID NO: 12); D58-BE4(SEQ ID NO: 16) 2 D56-AH7 (SEQ ID NO: 18); D13a-5 (SEQ ID NO: 20) 3D56-AG10 (SEQ ID NO: 22); D35-33 (SEQ ID NO: 24); D34-62 (SEQ ID NO: 26)4 D56-AA7 (SEQ ID NO: 28); D56-AE1 (SEQ ID NO: 30); 185-BD3 (SEQ ID NO:152) 5 D35-BB7 (SEQ ID NO: 32); D177-BA7 (SEQ ID NO: 34); D56A-AB6 (SEQID NO: 36); D144-AE2 (SEQ ID NO: 38) 6 D56-AG11 (SEQ ID NO: 40);D179-AA1 (SEQ ID NO: 42) 7 D56-AC7 (SEQ ID NO: 44); D144-AD1 (SEQ ID NO:46) 8 D144-AB5 (SEQ ID NO: 48) 9 D181-AB5 (SEQ ID NO: 50); D73-AC9 (SEQID NO: 52) 10 D56-AC12 (SEQ ID NO: 54) 11 D58-AB9 (SEQ ID NO: 56);D56-AG9 (SEQ ID NO: 58); D56-AG6 (SEQ ID NO: 60); D35-BG11 (SEQ ID NO:62); D35-42 (SEQ ID NO: 64); D35-BA3 (SEQ ID NO: 66); D34-57 (SEQ ID NO:68); D34-52 (SEQ ID NO: 70); D34-25 (SEQ ID NO: 72) 12 D56-AD10 (SEQ IDNO: 74) 13 56-AA11 (SEQ ID NO: 76) 14 D177-BD5 (SEQ ID NO: 78); D177-BD7(SEQ ID NO: 92) 15 D56A-AG10 (SEQ ID NO: 80); D58-BC5 (SEQ ID NO: 82);D58-AD12 (SEQ ID NO: 84) 16 D56-AC11 (SEQ ID NO: 86); D35-39 (SEQ ID NO:88); D58-BH4 (SEQ ID NO: 90); D56-AD6 (SEQ ID NO: 96) 17 D73A-AD6 (SEQID NO: 98); D70A-BA11 (SEQ ID NO: 100) 18 D70A-AB5 (SEQ ID NO: 104);D70A-AA8 (SEQ ID NO: 106) 19 D70A-AB8 (SEQ ID NO: 108); D70A-BH2 (SEQ IDNO: 110); D70A-AA4 (SEQ ID NO: 112) 20 D70A-BA1 (SEQ ID NO: 114);D70A-BA9 (SEQ ID NO: 116) 21 D70A-BD4 (SEQ ID NO: 118) 22 D181-AC5 (SEQID NO: 120); D144-AH1 (SEQ ID NO: 122); D34-65 (SEQ ID NO: 124) 23D35-BG2 (SEQ ID NO: 126) 24 D73A-AH7 (SEQ ID NO: 128) 25 D58-AA1 (SEQ IDNO: 130); D185-BC1 (SEQ ID NO: 142); D185-BG2 (SEQ ID NO: 144) 26D73-AE10 (SEQ ID NO: 132) 27 D56-AC12 (SEQ ID NO: 134) 28 D177-BF7 (SEQID NO: 136); D185-BE1 (SEQ ID NO: 146); D185-BD2 (SEQ ID NO: 148) 29D73A-AG3 (SEQ ID NO: 138) 30 D70A-AA12 (SEQ ID NO: 140); D176-BF2 (SEQID NO: 94) 31 D176-BC3 (SEQ ID NO: 154) 32 D176-BB3 (SEQ ID NO: 156) 33D186-AH4 (SEQ ID NO: 14)

GeneChip® microarray hybridization (Affymetrix Inc.; Santa Clara,Calif.) was used to identify genes with differential expression patternsbetween the converter and nonconverter near isogenic lines followingethylene activation. The chip size was 18 micron and the array formatwas 100-2187, accommodating 528 probe sets (11,628 probes). Seven pairsof hybridization were used to obtain independent verification ofmicroarray results. These consisted of one pair (converter/nonconverter)of 4407-33/4407-25 non-treated Burley tobacco samples, four pairs ofethylene treated 4407-33/4407-25 samples, one pair of ethylene treateddark tobacco NL Madole/181, another pair of lines near isogenic fornicotine conversion, and one pair of naturally senesced leaves of4407=33/25 (Table 3B).

TABLE 3B Converter:nonconverter normalized signal ratios from GeneChip ®Hybridization Ethylene Treated Untreated Ethylene Treated Burley DarkSenescence Burley (4407-33/25) (178/NL Burley (4407-33/25) Exp 1 Exp2Exp 3 Exp4 Madole) (4407-33/25) Induced D121-AA8 1.03 2.143 12.90 5.1712.19 16.60 2.57 D120-AH4 1.44 1.90 12.74 2.87 7.55 8.17 1.69 D35-BG111.73 2.32 13.06 22.22 19.10 28.76 3.40 Control Actin-Like I (5′) 1.180.99 0.74 0.73 0.57 1.02 0.97 Actin-Like I (3′) 1.09 1.12 0.81 1.08 0.790.93 0.85

All 14 sets of hybridizations were successful as evidenced by theExpression Report generated using detection instruments by GenomeExplorations, Inc. (Memphis, Tenn.). The main reports included analysesof Noise, Scale factor, background, total probe sets; number andpercentage of present and absent probe sets, signal intensity ofhousekeeping controls. The data were subsequently analyzed and presentedusing software GCOS in combination with other software. Signalcomparisons between treatment pairs were made, and overall data for allrespective probes for all hybridizations were compiled and theexpression data were also analyzed. Results based on the signalintensities showed that only two genes, D121-AA8 and D120-AH4 and onefragment, D35-BG11, which is a partial fragment of D121-AA8, hadreproducible induction in ethylene-treated converter lines when comparedto non-converter lines. The signal of a gene in a converter line, forexample, Burley tobacco variety 4407-33, was determined as the ratio tothe signal of a gene in a related non-converter isogenic line, 4407-25.Without ethylene treatment, the ratio of converter to non-convertersignals for all genes approached 1.00. To eliminate the influence ofbackground differences, normalized signal ratios were also calculated.Normalized signal ratios are obtained by dividing the treated pair ratiowith the corresponding non-pair ratio. Upon ethylene treatment andanalysis, it was determined that two genes, D121-AA8 and D120-AH4, wereinduced in converter lines relative to non-converter lines as determinedby four independent analyses. These two genes share 99.8% relativehomology and their relative hybridization signals in converter varietiesranged from approximately 2 to 22 fold higher than the signals in theirnon-converter counterparts. Based on the normalized ratios, twoactin-like, internal control clones, were not induced in converterlines. In addition, a fragment (D35-BG11), whose coding region isentirely contained in both the D121-AA8 and D120-AH4 genes, was highlyinduced in the same samples of paired isogenic converter andnonconverter lines. Furthermore, D121-AA8 and D120-AH4 genes werestrongly induced in converter lines of isogenic dark tobacco pairs, NLMadole and 181 (8 to 28 fold), thus demonstrating that ethyleneinduction of these genes in converter lines was an in planta response.The same genes were identified in the comparisons made fromhybridizations of naturally senesced samples of 4407-33/25 as well.RT-PCR assays of these materials using primers specific for D121-AA8verified the microarray results for this gene.

Based on these results, the D121-AA8 gene (the cDNA sequence of which isthe sequence of SEQ ID NO: 5; FIG. 4) was identified as the tobacconicotine demethylase gene of interest. In view of the p450 nomenclaturerule, it was determined that D121-AA8 is most similar to the p450s inthe CYP82E family (The Arabidopsis Genome Initiative (AGI) and TheArabidopsis Information Resource (TAIR); Frank, Plant Physiol. 110:1035-1046, 1996; Whitbred et al., Plant Physiol. 124:47-58, 2000);Schopfer and Ebel, Mol. Gen. Genet. 258:315322, 1998; and Takemoto etal., Plant Cell Physiol. 40:1232-1242, 1999).

Example 10 Biochemical Analysis of Enzymatic Activity

Biochemical analysis, for example, as described in previously filedapplications that are incorporated herein by reference, determined thatthe sequence of SEQ ID NO: 5 encodes a tobacco nicotine demethylase (SEQID NO:3).

In particular, the function of candidate clone D121-AA8 was confirmed asthe coding gene for nicotine demethylase, by assaying enzyme activity ofheterologously expressed p450 in yeast cells as follows.

1. Construction of Yeast Expression Vector

The putative protein-coding sequence of the tobacco nicotinedemethylase-encoding cDNA (D121-AA8), D120-AH4, D121-AA8, 208-AC-8, andD208-AD9, were cloned into the yeast expression vector pYeDP60.Appropriate BamHI and MfeI sites (underlined below) were introduced viaPCR primers containing these sequences either upstream of thetranslation start codon (ATG) or downstream of the stop codon (TAA). TheMfeI on the amplified PCR product is compatible with the EcoRI site onthe vector. The primers used to amplify the D121-AA8 cDNA were5′-TAGCTACGCGGATCCATGCTTTCTCCCATAGAAGCC-3′ (SEQ ID NO: 2194) and5′-CTGGATCACAATTGTTAGTGATGGTGATGGTGATGCGATCCTCTATAAAGCTC AGGT GCCAGGC-3′(SEQ ID NO: 2297). A segment of sequence coding nine extra amino acidsat the C-terminus of the protein, including six histidines, wasincorporated into the reverse primer to facilitate expression of 6-Histagged p450 upon induction. PCR products were ligated into pYeDP60vector after enzyme digestions in the sense orientation with referenceto the GAL10-CYC1 promoter. Proper construction of the yeast expressionvectors was verified by restriction enzyme analysis and DNA sequencing.In addition, expression of the p450 proteins was visualized on SDS-PAGEgel electrophoresis for the detergent phase of the yeast microsomes. Thepredicted size of the p450 proteins is 59 kD, based on the genesequence; a result that was confirmed by the gel analysis.

2. Yeast Transformation

The WAT11 yeast line, modified to express Arabidopsis NADPH-cytochromep450 reductase ATR1, was transformed with the pYeDP60-p450 cDNAplasmids. Fifty micro-liters of WAT11 yeast cell suspension was mixedwith ˜1 μg plasmid DNA in a cuvette with 0.2-cm electrode gap. One pulseat 2.0 kV was applied by an Eppendorf electroporator (Model 2510). Cellswere spread onto SGI plates (5 g/L bactocasamino acids, 6.7 g/L yeastnitrogen base without amino acids, 20 g/L glucose, 40 mg/LDL-tryptophan, 20 g/L agar). Transformants were confirmed by PCRanalysis performed directly on randomly selected colonies.

3. p450 Expression in Transformed Yeast Cells

Single yeast colonies were used to inoculate 30 mL SGI media (5 g/Lbactocasamino acids, 6.7 g/L yeast nitrogen base without amino acids, 20g/L glucose, 40 mg/L DL-tryptophan) and grown at 30° C. for about 24hours. An aliquot of this culture was diluted 1:50 into 1000 mL of YPGEmedia (10 g/L yeast extract, 20 g/L bacto peptone, 5 g/L glucose, 30ml/L ethanol) and grown until glucose was completely consumed asindicated by the colorimetric change of a Diastix urinalysis reagentstrip (Bayer, Elkhart, Ind.). Induction of cloned P450 was initiated byadding DL-galactose to a final concentration of 2%. The cultures weregrown for an additional 20 hours before used for in vivo activity assayor for microsome preparation.

WAT11 yeast cells expressing pYeDP60-CYP71D20 (a p450 catalyzing thehydroxylation of 5-epi-aristolochene and 1-deoxycapsidiol in Nicotianatabacum) were used as control for the p450 expression and enzymeactivity assays.

To evaluate the effectiveness of the yeast expression of the p450 ingreat detail, reduced CO difference spectroscopy was performed. Thereduced CO spectrum exhibited a peak at 450 nm proteins from all fourp450 transformed yeast lines. No similar peaks were observed in controlmicrosomes derived from control, untransformed yeast cells or the blank,vector control yeast cells. The results indicated that p450 proteinswere expressed effectively in yeast lines harboring the pYeDP60-CYP 450.Concentrations of expressed p450 protein in yeast microsome ranged from45 to 68 nmole/mg of total protein.

4. In Vivo Enzyme Assay

The nicotine demethylase activity in the transformed yeast cells wereassayed by feeding of yeast culture with DL-Nicotine(Pyrrolidine-2-¹⁴C). ¹⁴C labeled nicotine (54 mCi/mmol) was added to 75μl of the galactose-induced culture for a final concentration of 55 μM.The assay culture was incubated with shaking in 14 ml polypropylenetubes for 6 hours and was extracted with 900 μl methanol. Afterspinning, 20 μl of the methanol extract was separated with an rp-HPLCand the nornicotine fraction was quantitated by LSC.

The control culture of WAT11 (pYeDP60-CYP71D20) did not convert nicotineto nornicotine, showing that the WAT11 yeast strain does not containendogenous enzyme activities that can catalyze the step of nicotinebioconversion to nornicotine. In contrast, yeast expressing the tobacconicotine demethylase gene produced detectable amount of nornicotine,indicating the nicotine demethylase activity of the translation productof SEQ ID NO: 4 or SEQ ID NO: 5.

5. Yeast Microsome Preparation

After induction by galactose for 20 hours, yeast cells were collected bycentrifugation and washed twice with TES-M buffer (50 mM Tris-HCl, pH7.5, 1 mM EDTA, 0.6 M sorbitol, 10 mM 2-mercaptoethanol). The pellet wasresuspended in extraction buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.6M sorbitol, 2 mM 2-mercaptoethanol, 1% bovine serum album, ProteaseInhibitor Cocktail (Roche) at 1 tablet/50 ml). Cells were then brokenwith glass beads (0.5 mm in diameter, Sigma) and the cell extract wascentrifuged for 20 min at 20,000×g to remove cellular debris. Thesupernatant was subjected to ultracentrifugation at 100,000×g for 60 minand the resultant pellet contained the microsomal fraction. Themicrosomal fraction was suspended in TEG-M buffer (50 mM Tris-HCl, pH7.5, 1 mM EDTA, 20% glycerol and 1.5 mM 2-mercaptoethanol) at proteinconcentration of 1 mg/mL. Microsomal preparations were stored in aliquid nitrogen freezer until use.

6. Enzyme Activity Assay in Yeast Microsomal Preparations

Nicotine demethylase activity assays with yeast microsomal preparationswere performed. In particular, DL-Nicotine (Pyrrolidine-2-¹⁴C) wasobtained from Moravek Biochemicals and had a specific activity of 54mCi/mmol. Chlorpromazine (CPZ) and oxidized cytochrome c (cyt. C), bothP450 inhibitors, were purchased from Sigma. The reduced form ofnicotinamide adenine dinucleotide phosphate (NADPH) is the typicalelectron donor for cytochrome P450 via the NADPH:cytochrome P450reductase. NADPH was omitted for control incubation. The routine enzymeassay included microsomal proteins (around 1 mglrnl), 6 mM NADPH, and 55μM ¹⁴C labeled nicotine. The concentration of CPZ and Cyt. C, when used,was 1 mM and 100 μM, respectively. The reaction was carried at 25° C.for 1 hour and was stopped with the addition of 300 μM methanol to each25 μl reaction mixture. After centrifugation, 20 μl of the methanolextract was separated with a reverse-phase High Performance LiquidChromatography (HPLC) system (Agilent) using an Inertsil ODS-3 3μ(150×4.6 mm) chromatography column from Varian. The isocratic mobilephase was the mixture of methanol and 50 mM potassium phosphate buffer,pH 6.25, with ratio of 60:40 (v/v) and the flow rate was 1 ml/min. Thenornicotine peak, as determined by comparison with authentic non-labelednornicotine, was collected and subjected to 2900 tri-carb LiquidScintIDation Counter (LSC) (Perkin Elmer) for quantification. Theactivity of nicotine demethylase is calculated based on the productionof ¹⁴C labeled nornicotine over 1 hour incubation.

p450-like activity was observed in microsomal preparations from controlyeast cells expressing CYP71D₂₀ and the three test p450 yeast culturestransformed with the genes D120-AH4, D208-AC8, and D208-AD9. However,the control and the three test p450s did not show any nornicotineconversion formation suggesting that they did not contain an endogenousor induced enzyme that can catalyze the demethylation of nicotine. Incontrast, results from HPLC and LSC analyses showed detectablequantities of nornicotine produced from the demethylation of nicotineusing microsomal samples obtained from yeast cells expressing thetobacco nicotine demethylase gene (D121-AA8). These results indicatethat the nicotine demethylase activity results from the D121-AA8 geneproduct. The nicotine demethylase activity required NADPH and was shownto be inhibited by p450 specific inhibitors, consistent with tobacconicotine demethylase being a p450. The enzyme activity for tobacconicotine demethylase (D121-AA8) was approximately 10.8 pKat/mg proteinas calculated by radioactive intensity and protein concentrations. Atypical set of enzyme assay results obtained for the yeast cells isshown in the following table (Table 4).

TABLE 4 DEMETHYLASE ACTIVITY IN MICROSOMES OF YEAST CELLS EXPRESSINGDI21′-AA8 AND CONTROL P450 GENES Microsomes + Microsomes + 1 mM 100 μMMicrosomes − Sample Microsomes chlorpromazine cytochrome C NADPHD121-AA8 10.8 ± 1.2* pkat/mg 1.4 ± 1.3 pkat/mg 2.4 ± 0.7 pkat/mg proteinprotein protein Control Not Detected Not Detected Not Detected NotDetected (CYP71D20) *n = 12, others n = 3

Omission of NADPH from the assay using microsomes derived from D121-AA8yeast cells resulted in the abolishment of nicotine demethylaseactivity; hence no nornicotine was formed (Table 4). When two known P450inhibitors, Chlorpromazine (CPZ, 1 mM) and oxidized cytochrome c (cyt C,100 μM) were added into the enzyme assay mixtures separately andincubated for 1 hour before adding the methanol stop solution, nicotinedemethylase activities were decreased significantly (Table 4). Togetherthese experiments demonstrated that D121-AA8 encodes a cytochrome p450protein that catalyzes the conversion of nicotine to nornicotine whenexpressed in yeast.

Example 11 Related Amino Acid Sequence Identity of Isolated Nucleic AcidFragments

The amino acid sequences of nucleic acid sequences obtained forcytochrome p450 fragments from Example 8 were deduced. The deducedregion corresponded to the amino acid immediately after the GXRXCP(NG)(SEQ ID NO: 2273) sequence motif to the end of the carboxyl-terminus, orstop codon. Upon comparison of sequence identity of the fragments, aunique grouping was observed for those sequences with 70% amino acididentity or greater. A preferred grouping was observed for thosesequences with 80% amino acid identity or greater, more preferred with90% amino acid identity or greater, and a most preferred grouping forthose sequences 99% amino acid identity of greater. Several of theunique nucleic acid sequences were found to have complete amino acididentity to other fragments and therefore only one member with theidentical amino acid was reported.

The amino acid identity for Group 19 of Table 5 corresponded to threedistinct groups based on their nucleic acid sequences. The amino acidsequence of group members and their identity is shown in FIG. 5H. Theamino acid differences are indicated.

At least one member of each amino acid identity group was selected forgene cloning and functional studies using plants. In addition, groupmembers that are differentially affected by ethylene treatment or otherbiological differences as assessed by Northern and Southern analysiswere selected for gene cloning and functional studies. To assist in genecloning, expression studies and whole plant evaluations, peptidespecific antibodies can be prepared based on sequence identity anddifferential. sequence.

TABLE 5 Nicotiana p450 Amino Acid Sequence Identity Groups GROUPFRAGMENTS 1 D58-BG7 (SEQ ID NO: 11), D58-ABI (SEQ ID NO: 13) 2 D58-BE4(SEQ ID NO: 17) 3 D56-AH7 (SEQ ID NO: 19); D13a-5 (SEQ ID NO: 21) 4D56-AG10 (SEQ ID NO: 23); D34-62 (SEQ ID NO: 27) 5 D56-AA7 (SEQ ID NO:29); D56-AE1 (SEQ ID NO: 31); 185-BD3 (SEQ ID NO: 153) 6 D35-BB7 (SEQ IDNO: 33); D177-BA7 (SEQ ID NO: 35); D56A-AB6 (SEQ ID NO: 37); D144-AE2(SEQ ID NO: 39) 7 D56-AG11 (SEQ ID NO: 41); D179-AA1 (SEQ ID NO: 43) 8D56-AC7 (SEQ ID NO: 45); D144-AD1 (SEQ ID NO: 47) 9 D144-AB5 (SEQ ID NO:49) 10 D181-AB5 (SEQ ID NO: 51) D73-AC9 (SEQ ID NO: 53) 11 D56-AC12 (SEQID NO: 55) 12 D58-AB9 (SEQ ID NO: 57); D56-AG9 (SEQ ID NO: 59); D56-AG6(SEQ ID NO: 61); D35-BG11 (SEQ ID NO: 63); D35-42 (SEQ ID NO: 65);D35-BA3 (SEQ ID NO: 67); D34-57 (SEQ ID NO: 69); D34-52 (SEQ ID NO: 71)13 D56-AD10 (SEQ ID NO: 75) 14 D56-AA11 (SEQ ID NO: 77) 15 D177-BD5 (SEQID NO: 79); D177-BD7 (SEQ ID NO: 93) 16 D56A-AG10 (SEQ ID NO: 81);D58-BC5 (SEQ ID NO: 83); D58-AD12 (SEQ ID NO: 85) 17 D56-AC11 (SEQ IDNO: 87); D56-AD6 (SEQ ID NO: 97) 18 D73A-AD6 (SEQ ID NO: 99) 19 D70A-AB5(SEQ ID NO: 105); D70A-AB8 (SEQ ID NO: 109); D70A-BH2 (SEQ ID NO: 111);D70A-AA4 (SEQ ID NO: 113); D70A-BA1 (SEQ ID NO: 115); D70A-BA9 (SEQ IDNO: 117) 20 D70A-BD4 (SEQ ID NO: 119) 21 D181-AC5 (SEQ ID NO: 121);D144-AH1 (SEQ ID NO: 123); D34-65 (SEQ ID NO: 125) 22 D35-BG2 (SEQ IDNO: 127) 23 D73A-AH7 (SEQ ID NO: 129) 24 D58-AA1 (SEQ ID NO: 131);DI85-BC1 (SEQ ID NO: 143); D185-BG2 (SEQ ID NO: 145) 25 D73-AE10 (SEQ IDNO: 133) 26 D56-AC12 (SEQ ID NO: 135) 27 D177-BF7 (SEQ ID NO: 137);185-BD2 (SEQ ID NO: 149) 28 D73A-AG3 (SEQ ID NO: 139) 29 D70A-AA12 (SEQID NO: 141); D176-BF2 (SEQ ID NO: 95) 30 D176-BC3 (SEQ ID NO: 155) 31D176-BB3 (SEQ ID NO: 157) 32 D186-AH4 (SEQ ID NO: 15)

Example 12 Related Amino Acid Sequence Identity of Full-Length Clones

The nucleic acid sequences of full-length Nicotiana genes cloned inExample 5 were deduced for their entire amino acid sequence. Cytochromep450 genes were identified by the presence of three conserved p450domain motifs, which corresponded to UXXRXXZ (SEQ ID NO: 2274), PXRFXF(SEQ ID NO: 2275) or GXRXC (SEQ ID NO: 2276) at the carboxylterminuswhere U is E or K, X is any amino acid and Z is P, T, S or M. All p450genes were characterized for amino acid identity using a BLAST programcomparing their full-length sequences to each other and to known tobaccogenes. The program used the NCBI special BLAST tool (Align two sequences(b12seq), ncbi.nlm.nih.govlblast/bI2seqIb12.html on the World Wide Web).Two sequences were aligned under BLASTN without filter for nucleic acidsequences and BLASTP for amino acid sequences. Based on their percentageamino acid identity, each sequence was grouped into identity groupswhere the grouping contained members that shared at least 85% identitywith another member. A preferred grouping was observed for thosesequences with 90% amino acid identity or greater, a more preferredgrouping had 95% amino acid identity or greater, and a most preferredgrouping had those sequences 99% amino acid identity or greater. Usingthese criteria, 25 unique groups were identified and are depicted inTable 6. The amino acid sequence of the full-length nicotine demethylasegene was deduced to have the sequence provided in SEQ ID NO: 5.

Within the parameters used for Table 6 for amino acid identity, threegroups were found to contain greater than 85% or greater identity toknown tobacco genes. Members of Group 5 had up to 96% amino acididentity for the full-length sequence to GenBank sequence GI: 14423327(SEQ ID NO: 2271) (or AAK62346). Group 23 had up to 93% amino acididentity to GI: 14423328 (SEQ ID NO: 2277) (or AAK62347) and Group 24had 92% identity to GI: 14423318 (SEQ ID NO: 2278) (or AAK62343).

TABLE 6 Amino Acid Sequence Identity Groups of Full-Length Nicotianap450 Genes 1 D208-AD9 (SEQ ID NO: 233); D120-AH4 (SEQ ID NO: 189);D121-AA8 (SEQ ID NO: 191), DI22-AF10 (SEQ ID NO: 193); D103-AH3 (SEQ IDNO: 231); D208-AC8 (SEQ ID NO: 227); D235-AB1 (SEQ ID NO: 255) 2D244-AD4 (SEQ ID NO: 259); D244-AB6 (SEQ ID NO: 283); D285-AA8 (SEQ IDNO: 2205); D285-AB9 (SEQ ID NO: 2206); D268-AE2 (SEQ ID NO: 279). 3D100A-AC3 (SEQ ID NO: 177); D100A-BE2 (SEQ ID NO: 2209) 4 D205-BE9 (SEQID NO: 285); D205-BG9 (SEQ ID NO: 211); D205-AH4(SEQ ID NO: 303) 5D259-AB9 (SEQ ID NO: 269); D257-AE4 (SEQ ID NO: 277); D147-AD3 (SEQ IDNO: 203) 6 D249-AE8 (SEQ ID NO: 265); D-248-AA6 (SEQ ID NO: 263) 7D233-AG7 (SEQ ID NO: 275); D224-BD11 (SEQ ID NO: 249); DAF10 8 D105-AD6(SEQ ID NO: 181); D215-AB5 (SEQ ID NO: 229); D135-AE1 (SEQ ID NO: 199) 9D87A-AF3 (SEQ ID NO: 225), D210-BD4 (SEQ ID NO: 273) 10 D89-AB1 (SEQ IDNO: 159); D89-AD2 (SEQ ID NO: 161); D163-AG11 (SEQ ID NO: 207);D163-AF12 (SEQ ID NO: 205) 11 D267-AFI0 (SEQ ID NO: 305); D96-AC2 (SEQID NO: 169); D96:-AB6 (SEQ ID NO: 167); D207-AA5 (SEQ ID NO: 213);D207-AB4 (SEQ ID NO: 215); D207- AC4 (SEQ ID NO: 217) 12 D98-AG1 (SEQ IDNO: 173); D98-AA1 (SEQ ID NO: 171) 13 D209-AA12 (SEQ ID NO: 221);D209-AA11; D209-AH10 (SEQ ID NO: 223); D209-AH12 (SEQ ID NO: 241);D90A-BB3 (SEQ ID NO: 163) 14 D129-AD10 (SEQ ID NO: 197); D104A-AE8 (SEQID NO: 179) 15 D228-AH8 (SEQ ID NO: 253); D228-AD7 (SEQ ID NO: 251),D250-AC11 (SEQ ID NO: 267); D247-AH1 (SEQ ID NO: 261) 16 D128-AB7 (SEQID NO: 195); D243-AA2 (SEQ ID NO: 257); D125-AF11 (SEQ ID NO: 237) 17D284-AH5 (SEQ ID NO: 307); D110-AF12 (SEQ ID NO: 185) 18 D221-BB8 (SEQID NO: 243) 19 D222-BH4 (SEQ ID NO: 245) 20 D134-AE11 (SEQ ID NO: 239)21 D109-AH8 (SEQ ID NO: 183) 22 D136-AF4 (SEQ ID NO: 287) 23 D237-AD1(SEQ ID NO: 235) 24 D112-AA5 (SEQ ID NO: 187) 25 D283-AC1 (SEQ ID NO:281)

The full-length genes were further grouped based on the highly conservedamino acid homology between UXXRXXZ p450 domain (SEQ ID NO: 2274) andGXRXC p450 domain (SEQ ID NO: 2276) near the end the carboxyl-terminus.As shown in FIGS. 160A to 160E, individual clones were aligned based onthe sequence homology between the conserved domains and placed indistinct identity groups. In several cases, although the nucleic acidsequence of the clone was unique, the amino acid sequence for the regionwas identical. The preferred grouping was observed for those sequenceswith 90% amino acid identity or greater, a more preferred group had 95%amino acid identity or greater, and a most preferred grouping had thosesequences 99% amino acid identity of greater. The final grouping wassimilar to that based on the percent identity for the entire amino acidsequence of the clones except for Group 17 (of Table 6) which wasdivided into two distinct groups.

Within the parameters used for amino acid identity in Table 7, threegroups were found to contain 90% or greater identity to known tobaccogenes. Members of Group 5 had up to 93.4% .amino acid identity for fulllength sequences to the GenBank sequence of GI: 14423326 (SEQ ID NO:2279) (or AAK62346). Group 23 had up to 91.8% amino acid identity to GI:14423328 (SEQ ID NO:2277) (or AAK62347) and Group 24 had 98.8% identityto GI: 14423318 (SEQ ID NO: 2278) (or AAK62342).

TABLE 7 Amino Acid Sequence Identity Groups of Regions between ConservedDomains of Nicotiana p450 Genes 1 D208-AD9 (SEQ ID NO: 233); D120-AH4(SEQ ID NO: 189); D121-AA8 (SEQ ID NO: 191), D122-AF10 (SEQ ID NO: 193);D103-AH3 (SEQ ID NO: 231); D208-AC8 (SEQ ID NO: 227); D235-AB1 (SEQ IDNO: 255) 2 D244-AD4 (SEQ ID NO: 259); D244-AB6 (SEQ ID NO: 283);D285-AA8 (SEQ ID NO: 2205); D285-AB9 (SEQ ID NO: 2206); D268-AE2 (SEQ IDNO: 279) 3 D100A-AC3 (SEQ ID NO: 177); D100A-BE2 (SEQ ID NO: 2209) 4D205-BE9 (SEQ ID NO: 285); D205-BG9 (SEQ ID NO: 211); D205-AH4 (SEQ IDNO: 303) 5 D259-AB9 (SEQ ID NO: 269); D257-AE4 (SEQ ID NO: 277);D147-AD3 (SEQ ID NO: 203) 6 D249-AE8 (SEQ ID NO: 265); D-248-AA6 (SEQ IDNO: 263) 7 D233-AG7 (SEQ ID NO: 275); D224-BD11 (SEQ ID NO: 249); DAF108 D105-AD6 (SEQ ID NO: 181); D215-AB5 (SEQ ID NO: 229); D135-AE1 (SEQ IDNO: 199) 9 D87A-AF3 (SEQ ID NO: 225), D210-BD4 (SEQ ID NO: 273) 10D89-AB1 (SEQ ID NO: 159); D89-AD2 (SEQ ID NO: 161); DI63-AG11 (SEQ IDNO: 207); D163-AF12 (SEQ ID NO: 205) 11 D267-AF10 (SEQ ID NO: 305);D96-AC2 (SEQ ID NO: 169); D96-AB6 (SEQ ID NO: 167); D207-AA5 (SEQ ID NO:213); D207-AB4 (SEQ ID NO: 215); D207-AC4 (SEQ ID NO: 217) 12 D98-AG1(SEQ ID NO: 173); D98-AA1 (SEQ ID NO: 171) 13 D209-AA12 (SEQ ID NO:221); D209-AA11; D209-AH10 (SEQ ID NO: 223); D209-AH12 (SEQ ID NO: 241);D90A-BB3 (SEQ ID NO: 163) 14 DI29-AD10 (SEQ ID NO: 197); D104A-AE8 (SEQID NO: 179) 15 D228-AH8 (SEQ ID NO: 253); D228-AD7 (SEQ ID NO: 251),D250-AC11 (SEQ ID NO: 267); D247-AH1 (SEQ ID NO: 261) 16 D128-AB7 (SEQID NO: 195); D243-AA2 (SEQ ID NO: 257); DI25-AF11 (SEQ ID NO: 237) 17D284-AH5 (SEQ ID NO: 307); D110-AF12 (SEQ ID NO: 185) 18 D221-BB8 (SEQID NO: 243) 19 D222-BH4 (SEQ ID NO: 245) 20 D134-AE11 (SEQ ID NO: 239)21 D109-AH8 (SEQ ID NO: 183) 22 D136-AF4 (SEQ ID NO: 285) 23 D237-AD1(SEQ ID NO: 235) 24 D112-AA5 (SEQ ID NO: 187) 25 D283-AC1 (SEQ ID NO:281) 26 D110-AF12 (SEQ ID NO: 185)

Example 13 Nicotiana Cytochrome P450 Clones Lacking One or More of theTobacco P450 Specific Domains

Four clones had high nucleic acid homology, ranging 90% to 99% nucleicacid homology, to other tobacco cytochrome genes reported in Table 6.The four clones included D136-AD5 (SEQ ID NO: 292), D138-AD12 (SEQ IDNO: 294), D243-AB3 (SEQ ID NO: 298) and D250-AC11 (SEQ ID NO: 300).However, due to a nucleotide frameshift, these genes did not contain oneor more of three C-terminus cytochrome p450 domains and were excludedfrom identity groups presented in Table 6 or Table 7.

The amino acid identity of one clone, D95-AG1, did not contain the thirddomain, GXRXC (SEQ ID NO: 2276), used to group p450 tobacco genes inTable 6 or Table 7. The nucleic acid sequence of this clone had lowhomology to other tobacco cytochrome genes and, therefore, this clonerepresents a novel group of cytochrome p450 genes in Nicotiana.

Example 14 Use of Nicotiana Cytochrome P450 Fragments and Clones inAltered Regulation of Tobacco Qualities

The use of tobacco p450 nucleic acid fragments or whole genes are usefulin identifying and selecting those plants that have altered tobaccophenotypes or tobacco constituents and, more importantly, alteredmetabolites. Transgenic tobacco plants are generated by a variety oftransformation systems that incorporate nucleic acid fragments orfull-length genes, selected from those reported herein, in orientationsfor either down-regulation, for example anti-sense orientation, orover-expression for example, sense orientation and the like. Forover-expression to full-length genes any nucleic acid sequence thatencodes the entire or a functional part or amino acid sequence of thefull-length genes described in this invention is desirable. Such nucleicacid sequences desirably are effective for increasing the expression ofa certain enzyme and thus resulting in phenotypic effect with Nicotiana.Nicotiana lines that are homozygous are obtained through a series ofbackcrossing and assessed for phenotypic changes including, but notlimited to, analysis of endogenous p450 RNA, transcripts, p450 expressedpeptides and concentrations of plant metabolites using techniquescommonly available to one having ordinary skill in the art. The changesexhibited in the tobacco plants provide information on the functionalrole of the selected gene of interest or are of use as preferredNicotiana plant species.

Example 15 Cloning of the Genomic Tobacco Nicotine Demethylase fromConverter Burley Tobacco

Genomic DNA was extracted from converter Burley tobacco plant line4407-33 (a Nicotiana tabacum variety 4407 line) using Qiagen Plant Easykit as described in above Examples (see also the manufacturer'sprocedure).

The primers were designed based on the 5′ promoter and 3′ UTR regioncloned in previous examples. The forward primers were 5′-GGC TCT AGA TAAATC TCT TAA GTT ACT AGG TTC TAA-3′ (SEQ ID NO: 2280) and 5′-TCT CTA AAGTCC CCT TCC-3′ (SEQ ID NO: 2288) and the reverse primers were 5′-GGC TCTAGA AGT CAA TTA TCT TCT ACA AAC CTT TAT ATA TTA GC-3′ (SEQ ID NO: 2281),and 5′-CCA GCA TTC CTC AAT TTC-3′ (SEQ ID NO: 2289). PCR was applied tothe 4407-33 genomic DNA with 100 μl of reaction mix. Pfx high fidelityenzyme was used for PCR amplification. The PCR product was visualized on1% agarose gel after electrophoresis. A single band with molecularweight of approximately 3.5 kb was observed and excised from the gel.The resulting band was purified using a gel purification kit (Qiagen;based on manufacturer's procedure). The purified DNA was digested byenzyme Xba 1 (NEB; used according to the manufacturer's instructions).The pBluescript plasmid was digested by Xba 1 using same procedure. Thefragment was gel purified and ligated to pBluescript plasmid. Theligation mix was transformed into competent cell GM 109 and plated ontoLB plate containing 100 mg/l of ampicillin with blue/white selection.The white colonies were picked and grown into 10 ml LB liquid mediacontaining ampicillin. The DNA was extracted by miniprep. The plasmidDNA containing the insert was sequenced using a CEQ 2000 sequencer(Beckman, Fullerton, Calif.) based on the manufacturer's procedure. TheT3 and T7 primers and 8 other internal primers were used for sequencing.The sequence was assembled and analyzed, thus providing the genomicsequence (SEQ ID NO: 4). Based on the genomic sequence, it wasdetermined that the nicotine demethylase gene in both converter andnonconverter tobacco lines does not contain a transposable element.

Comparison of the sequence of SEQ ID NO: 5 with the sequence of SEQ IDNO: 4 allowed the determination of a single intron within the codingportion of the gene (identified as the sequence of SEQ ID NO: 7). Asshown in FIG. 1, the genomic structure of the tobacco nicotinedemethylase includes two exons flanking a single intron. The first exonspans nucleotides 2010 to 2949 of SEQ ID NO: 4, which encodes aminoacids 1-313 of SEQ ID NO: 3, and the second exon spans nucleotides 3947to 4562 of SEQ ID NO: 4, which encode amino acids 314-517 of SEQ ID NO:3. Accordingly, the intron spans nucleotides 2950-3946 of SEQ ID NO: 4.The intron sequence is provided in SEQ ID NO: 7. The translation productof the genomic DNA sequence is provided in SEQ ID NO: 3. The tobacconicotine demethylase amino acid sequence contains an endoplasmicreticulum membrane anchoring motif.

Example 16 Cloning 5′ Flanking Sequences (SEQ ID NO:8) and 3′UTR (SEQ IDNO:9) from Converter Tobacco

A. Isolation of Total DNA from Converter Tobacco Leaves Tissue

Genomic DNA was isolated from leaves of converter tobacco 4407-33. Theisolation of DNA was performed using a DNeasy Plant Mini Kit from thecompany Qiagen, Inc. (Valencia, Calif.) according to the manufacturer'sprotocol. The manufacturer's manual Dneasy' Plant Mini and DNeasy PlantMaxi Handbook, Qiagen January 2004 is incorporated hereby as reference.The procedure for DNA preparation included the following steps: Tobaccoleaf tissue (approximately 20 mg dry weight) was ground to a fine powderunder liquid nitrogen for 1 minute. The tissue powder was transferredinto a 1.5 ml tube. Buffer AP1 (400 μl) and 4 μl of RNase stock solution(100 mg/ml) were added to a maximum of 100 mg of ground leaf tissue andvortexed vigorously. The mixture was incubated for 10 min at 65° C. andmixed 2-3 times during incubation by inverting tube. Buffer AP2 (130 μl)was then added to the lysate. The mixture was mixed and incubated for 5min on ice. The lysate was applied to a QIAshredder Mini Spin Column andcentrifuged for 2 min (14,000 rpm). The flow-through fraction wastransferred to a new tube without disturbing the cell-debris pellet.Buffer AP3/E (1.5 volumes) was then added to the cleared lysate andmixed by pipetting. The mixture (650 μl) from the preceding stepincluding any precipitate was applied to a DNeasy Mini Spin Column. Themixture was centrifuged for 1 min at >6000×g (>8000 rpm) and theflow-through was discarded. This was repeated with the remaining sampleand the flow-through and collection tube were discarded. DNeasy MiniSpin Column was placed in a new 2 ml collection tube. Then buffer AW(500 μl) was added to the DNeasy column and centrifuged for 1 min (>8000rpm). The flow-through was discarded. The collection tube was reused inthe next step. Buffer AW (500 μl) was then added to the DNeasy columnand centrifuged for 2 min (>14,000 rpm) in order to dry the membrane.The DNeasy column was transferred to a 1.5 ml tube. Then Buffer AE (100μl) was pipetted onto the DNeasy membrane. The mixture was incubated for5 min at room temperature (15-25° C.) and then centrifuged for 1 min(>8000 rpm) to elute.

The quality and quantity of the DNA was estimated by running samples onan agarose gel.

B. Cloning of 5′ Flanking Sequences of the Structural Gene

A modified inverse PCR method was used to clone 750 nucleotides of the5′ flanking sequences of the structural gene from SEQ ID NO: 5. First,appropriate restriction enzymes were selected based on the restrictionsite in the known sequence fragment and the restriction sites distancedownstream of the 5′ flanking sequences. Two primers were designed basedon this known fragment. The forward primer was located downstream of thereverse primer. The reverse primer was located in the 3′ portion of theknown fragment.

The cloning procedure included the following steps:

The purified genomic DNA (5 μl) was digested with 20-40 units of theappropriate restriction enzyme (EcoRI and SpeI) in a 50 μl reactionmixture. An agarose gel electrophoresis with a 1/10 volume of thereaction mixture was performed to determine if the DNA was digested tocompletion. A direct ligation was performed after thorough digestion byligating overnight at 4° C. A reaction mixture of 200 μl containing 10μl of digested DNA and 0.2 μl of T4 DNA ligase (NEB) was ligatedovernight at 4° C. PCR on the ligation reaction was performed after anartificial small circular genome was obtained. PCR was performed with 10μl of ligation reaction and 2 primers from known fragments in twodifferent directions in 50 μl reaction mixture. A gradient PCR programwith annealing temperatures of 45-56° C. was applied.

Agarose gel electrophoresis was performed to check the PCR reaction. Thedesired band was cut from the gel and a QIAquick gel purification Kitfrom QIAGEN was used to purify the band. The purified PCR fragments wereligated into a pGEM-T Easy Vector (Promega, Madison, Wis.) followingmanufacturer's instructions. The transformed DNA plasmids were extractedby miniprep using SV Miniprep kit (Promega, Madison, Wis.) following themanufacturer's instructions. Plasmid DNA containing the insert wassequenced using a CEQ 2000 sequencer (Beckman, Fullerton, Calif.).Approximately 758 nt (nucleotides 1241-2009 of SEQ ID NO:4) of the 5′flanking sequence were cloned by the method described above.

C. Cloning of the Longer 5′ Flanking Sequences (SEQ ID NO: 8) of theStructural Gene

BD GenomeWalker Universal Kit (Clontech laboratories, Inc., PaloAlto,Calif.) was used for cloning additional 5′ flanking sequence of thestructural gene, D121-AA8 according to the manufacturer's user manual.The manufacturer's manual BD GenomeWalker August, 2004 is incorporatedhereby as reference. The size and purity of tobacco genomic DNA weretested by running samples on a 0.5% agarose gel. A total of 4 blunt-endreactions (DRA I, STU I, ECOR V, PVU II) were set up for tobacco 33library genome walking construction. After purification of the digestedDNAs, the digested genomic DNAs were ligated to the genome walkeradaptor. Primary PCR reactions were applied to the four digested DNA'sby using adaptor primer API and the gene specific primer from D121-AA8(CTCTATTGATACTAGCTGGTTTTGGAC; SEQ ID NO: 2282). The primary PCR productswere used directly as templates for the nested PCR. The adaptor nestedprimer provided by the kit and the nested primer from the known cloneD121-AA8 (SEQ ID NO: 5) (GGAGGGAGAGTATAACTTACGGATTC; SEQ ID NO: 2283)were used in the PCR reaction. PCR products were checked by running gelelectrophoreses. The desired bands were sliced out from the gel, and thePCR fragments were purified using QIAquick gel purification Kit fromQIAGEN. The purified PCR fragments were ligated into a pGEM-T EasyVector (Promega, Madison, Wis.) following manufacturer's instructions.The transformed DNA plasmids were extracted by miniprep using the SVMiniprep kit (Promega, Madison, Wis.) and following the manufacturer'sinstructions. Plasmid DNA containing the insert was sequenced using aCEQ 2000 sequencer (Beckman, Fullerton, Calif.). Another approximately853 nt of the 5′ flanking sequence, including nucleotides 399-1240 ofSEQ ID NO: 4, were cloned by the method described above. The nucleicacid sequence of the 3′UTR region is set forth in SEQ ID NO:9

A second round of the genome walking was performed according to the samemethod with the difference that the following primers GWRIA(5′AGTAACCGATTGCTCACGTTATCCTC-3′) (SEQ ID NO: 2284) and GWR2A(5′CTCTATTCAACCCCACACGTAACTG-3′) (SEQ ID NO: 2285) were used. Anotherapproximately 398 nt of flanking sequence, including nucleotides 1-398of SEQ ID NO: 4, were cloned by this method.

A search for regulatory elements revealed that, in addition to “TATA”box, “CAAT” boxes, and “GAGA” boxes, several MYB-like recognition sitesand organ specificity elements are present in the tobacco nicotinedemethylase promoter region. Putative elicitor responsive elements andnitrogen-regulated elements, identified using standard methods, are alsopresent in the promoter region.

D. Cloning of 3′ Flanking Sequences of the Structural Gene

BD GenomeWalker Universal Kit (Clontech laboratories, Inc., PaloAlto,Calif.) was used for cloning of 3′ flanking sequence of the structuralgene, D121-AA8 according to the manufacturer's user manual. The cloningprocedure is the same as describes in the preceding Section C of thisexample, except for the gene specific primers. The first primer wasdesigned from close to the end of D121-AA8 structural gene (5′-CTA AACTCT GGT CTG ATC CTG ATA CTT-3′) (SEQ ID NO: 2286). The nested primer wasdesigned further downstream of primer 1 of the D121-AA8 structural gene(CTA TAC GTA AGG TAA ATC CTGTGG AAC) (SEQ ID NO: 2287). The final PCRproducts were checked by gel electrophoreses. The desired bands wereexcised from the gel. The PCR fragments were purified using QIAquick gelpurification Kit from QIAGEN. The purified PCR fragments were ligatedinto a pGEM-T Easy Vector (Promega, Madison, Wn following manufacturer'sinstructions. The transformed DNA plasmids were extracted by miniprepusing SV Miniprep kit (Promega, Madison, Wis.) following manufacturer'sinstructions. Plasmid DNA containing the insert was sequenced using aCEQ 2000 sequencer (Beckman, Fullerton, Calif.). Approximately 1617nucleotides of additional 3′ flanking sequence (nucleotides 4731-6347 ofSEQ ID NO: 4) were cloned by the method described above. The nucleicacid sequence of the 3′UTR region is shown in FIG. 7.

Example 17 Screening the Nicotiana Genus for the Presence or Absence ofa Nicotine Demethylase Gene

Forty-three Nicotiana species, forty-nine Nicotiana rustica lines, andapproximately six hundred Nicotiana tabacum lines were seeded in potsand the resulting plants were grown in the greenhouse as shown in Table8 below.

TABLE 8 Scientific Name or Common Inventory Name or Source NumberNicotiana africana TW6 Nicotiana amplexicaulis TW10 Nicotiana arentsiiTW12 Nicotiana attenuata TW13 Nicotiana benavidesii TW 15 Nicotianabenthamiana TW16 Nicotiana biRelovii TW18 Nicotiana bonariensis TW28Nicotiana clevelandii TW30 Nicotiana corymbosa TW35 Nicotiana debneyiTW36 Nicotiana excelsior TW46 Nicotiana exigua TW48 Nicotiana RlaucaTW53 Nicotiana glutinosa TW58 Nicotiana goodspeedii TW67 Nicotianagossei TW68 Nicotiana hesperis TW69 Nicotiana ingulba TW71 Nicotianakawakamii TW72 Nicotiana knightiana TW73 Nicotiana maritima TW82Nicotiana megalosiphon TW83 Nicotiana miersii TW85 Nicotiana nesophilaTW87 Nicotiana noctif[ora TW88 Nicotiana nudicaulis TW90 Nicotianaotophora TW94 Nicotiana palmeri TW98 Nicotiana paniculata TW99 Nicotianapetunioides TW105 Nicotiana plumbaginijolia TW106 Nicotiana repandaTW110 Nicotiana rosulata TW112 Nicotiana rotundifolia TW114 Nicotianarustica TW116 Nicotiana setchelli TW121 Nicotiana solanifolia TW123Nicotiana stocktonii TW126 Nicotiana eastii TW127 Nicotiana suaveolensTW128 Nicotiana thrysiflora TW139 Nicotiana tomentosa TW140 Nicotianatomentosiformis TW142 Nicotiana trigonophylla TW143 Nicotiana undulataTW145 4384-HHS TR1 43103-5 TR10 43104-1 TR11 4401 TR12 Brasilia #7 TR13Brasilia #23 TR14 Brasilia Selvaggio TR15 Brasilia TR16 Erbasanta TR1768 Olson TR18 C 39-′193 TR19 4385 L-5-6 TR2 German #2 TR20 German #1TR21 Mahorka #1 TR22 Mahorka #2 TR23 Mahorka #3 TR24 Mahorka #4 TR25Mahorka #5 TR26 Mahorka #6 TR27 Mahorka #7 TR28 Mahorka #8 TR29 4386L-5-6 TR3 Mahorka #9 TR30 Mahorka #10 TR31 Mahorka #11 TR32 Mahorka #12TR33 Kostoff TR34 Bak #46 TR35 Koriotes TR36 Jainkaya Sol TR37 Jainkayabl TR38 Drosqi TR39 4390 L-5-2-1 TR4 14 No. 23057 TR40 Edinburg 25 TR41Ja. Bot. Car. TR42 R. Bot. Car. TR43 HARBIN TR44 Normal TR45 Matsui TR46Buni TR47 DUMONT TR48 Chinensis TR49 4398 L-5-2-1 TR5 Campanulata TR50Acutifolia TR51 Fructicosa TR52 Acutifolia TR53 Nordugel TR54 GC-1 TR55Hasankeyf TR56 PNE 241-5 TR57 PNE 362-4 TR58 PNE 369-3 TR59 4399 L-5-2-1TR6 PNE 373-13 TR60 PNE 407-5 TR61 PNE 412-8 TR62 PNE 417-4 TR63 PNE418-6 TR64 PNE420-6 TR65 PNE 427-4 TR66 TI 1674 TR67 TI 1685 TR68 TI1686 TR69 43054 TR7 TI 1693 TR70 Rustica TR71 Rustica TR72 Rustica TR73Rustica TR74 Rustica TR75 Rustica TR76 Rustica TR77 Selection fromPI499194 TR78 Selection from PI499200 TR79 43101 TR8 Selection fromPI499206 TR80 93024 TR81 Rustica TR82 Florida 301 TC 195 DF300 TC 465Mos Res Black Mammoth TC 481 Tom Rossen (TR) Madole TC 486 MS KY 16 TC521 NC-BMR42 TC 570 Ntabacum KDH-926 TC 575 Ntabacum KDH-959 TC 576Ntabacum KDH-960 TC 577 Nance TC 616 TND94 TC 621 Burley Mammoth KY16TC12 Ex. 12 TC13 Golden Burley TC14 GR2 TC15 GR5 TC16 GR6 TC17 GR 13TC21 KY 153 TC216 KY 157 TC217 KY 163 TC219 GR 14 TC22 KY 165 TC220Little Sweet Orinoco TC221 Little Yellow TC222 Madole (NN) TC223 OneSucker TC224 Virginia 312 TC228 GR 17 TC23 GR 18 TC25 GR 19 TC26 GR 36TC28 GR 38 TC29 GR 38A TC30 GR 40 TC31 GR 42 TC32 GR 42C TC33 GR 43 TC34GR 44 TC35 GR 45 TC36 GR 53 TC39 Greenbrior TC40 H-47 TC42 Harouova TC43Harrow 12 TC44 Harrow Velvet TC45 Aurelius TC459 HarwID TC46 BlackMammoth TC460 Browleaf TC462 D-534-A-1 TC464 DF 516 TC467 DF 911 TC468HI Burley 21 TC47 Improved Madole TC471 Jernigan's Madole TC472 Kentucky151 TC473 Little Crittenden TC476 Lizard Tail Orinoco TC477 ImprovedBrior TC48 Mos Res (MR/NN) Madole TC480 Mos Res Little Crittenden TC482Mos Res Little Wood TC483 Narrow Leaf (NL) Madole TC484 Sears SpecialTC485 VA 310 TC487 Walkers Broadleaf TC489 Judy's Pride TC49 Woods TC490Baur TC491 Bel MS-1 TC492 Bel MS-2 TC493 Catterton TC494 Dean TC495Gertz TC496 Keller TC497 Maryland 10 TC498 Maryland 14 D2 TC499 KellyBrownleaf TC50 Maryland 21 TC500 Maryland 59 TC501 Maryland 64 TC502Maryland 201 TC503 Maryland 341 TC504 Maryland Stand-Up Mammoth TC508 MDB100 TC509 Kelly Burley TC51 Moore TC511 Posey TC512 Robinson MedBroadleaf TC513 Sweeney TC514 Thompson TC515 Ward TC516 Wilson TC517 MS400 TC518 MS4 02 TC519 KY 1 TC52 MS Burleyl TC520 MS PA Swarr HibshmanTC523 SB400 TC524 SB Burley 1 TC526 K5 TC53 Samsun TC536 Samsun (PHYB)-1TC537 Samsun (PHYB)-2 TC538 KY9 TC54 Samsun Holmes (NN) TC540 Samsun NO15 TC541 Samsun-BLK SHK Tol TC542 Smyrna TC543 Smyrna NO 9 TC544 SmyrnaNO 23 TC545 Smyrna-BLK SHK Tol TC546 Stanimaka NO 20 TC547 Turkish TC548Xanthi (Mitchell-Mor) TC549 Xanthi (Smith) TC550 Xanthi Yaka NO 18ATC552 Xanthi-Parental TC554 Perique TC556 KY12 TC56 VA 309 TC560 VA 409TC562 KYBSS TC565 NC-BMR 90 TC571 KDH-926 TC575 KDH-926 TC575 KDH-959TC576 KDH-959 TC576 KDH-960 TC577 C8 TC578 VA 331 TC592 Smith TO 448ATC594 LN KY 171 TC605 SI KY 171 TC607 SI KY 160 TC608 KY19 TC61 IG KY171 TC610 IG KY 160 TC611 PYKY 160 TC612 PY KY 171 TC613 Shirey TC617TND950 TC622 VA 355 TC638 VA 359 TC639 OS 802 TC640 Black Mammoth SMStalk TC641 Elliot Madole TC643 Goose Creek Red TC644 Little Wood TC645KY56 TC72 KY57 TC73 KY58 TC74 Uniform TC83 Warner TC86 Yellow Twist BudTC88 Venezuela TI 106 N. tabacum Hoja parado (Galpoa) TI 1068 ArgentinaTI 1068 Peru TI 1075 Turkey TI 1217 Turkey TI 1218 Turkey TI 1219 TurkeyTI 1222 Turkey TI 1223 Turkey TI 1224 Turkey TI 1225 Turkey TI 1229Turkey TI 1230 Turkey TI 1235 Turkey TI 1236 Turkey TI 1237 Spain TI1239 Spain TI 1245 Spain TI 1246 Spain TI 1247 Spain TI 1250 Spain TI1251 Spain TI1253 Yugoslavia TI 1254 Paraguay TI 1255 Ethiopia TI 1268Ethiopia TI 1269 Ethiopia TI 1270 Ethiopia TI 1271 Korea, South TI 1278Brazil TI 128 Korea, South TI 1280 Yugoslavia TI 1282 Yugoslavia TI 1283Yugoslavia TI 1284 Yugoslavia TI 1285 Yugoslavia TI 1286 Yugoslavia TI1287 Brazil TI 129 Yugoslavia TI 1291 Yugoslavia TI 1292 Yugoslavia TI1293 Yugoslavia TI 1295 Yugoslavia TI 1296 Yugoslavia TI 1297 Bolivia TI1301 Bolivia TI 1302 Argentina TI 1306 Papua New Guinea TI 1311 GreeceTI 1313 New Zealand TI 1315 New Zealand TI 1317 New Zealand TI 1318Yugoslavia TI 1320 Yugoslavia TI 1321 Yugoslavia TI 1322 Yugoslavia TI1324 Yugoslavia TI 1325 Yugoslavia TI 1326 Yugoslavia TI 1327 YugoslaviaTI 1329 Yugoslavia TI 1332 Yugoslavia TI 1333 Austria TI 1349 Cuba TI1373 Cuba TI 1375 Cuba TI 1376 Bulgaria TI 1378 Bulgaria TI 1379Bulgaria TI 1380 Bulgaria TI 1380 Bulgaria TI 1381 Bulgaria TI 1382Bulgaria TI 1383 Bulgaria TI 1384 Bulgaria TI 1385 Bulgaria TI 1386Bulgaria TI 1387 Bulgaria TI 1388 Bulgaria TI 1389 Bulgaria TI 1407Bulgaria TI 1408 Bulgaria TI 1409 Bulgaria TI 1410 Bulgaria TI 1411Bulgaria TI 1412 Italy TI 1414 Liberia TI 1426 Liberia TI 1427 Poland TI1444 Cuba TI 1452 Cuba TI 1453 Brazil TI 1455 Germany TI 1459 Germany TI1460 Spain TI 1485 Bulgaria TI 1492 Bulgaria TI 1493 Bulgaria TI 1494Bulgaria TI 1496 Switzerland TI 1506 Australia TI 1507 Australia TI 1508Germany TI 1532 Germany TI 1533 Belgium TI 1534 Belgium TI 1535 AustriaTI 1536 Italy TI 1538 Iran TI 1555 Iran TI 1556 United States TI 1561United States TI 1562 United States TI 1563 Poland TI 1567 Poland TI1568 Poland TI 1569 Poland TI 1570 Japan TI 158 Japan TI 1594 Italy TI1595 Italy TI 1596 Italy TI 1599 Italy TI 1600 Italy TI 1601 Italy TI1602 Rhodesia TI 1603 Japan TI 1604 Japan TI 1605 Yugoslavia TI 1623United States TI 186 United States TI 187 United States TI 240 UnitedStates TI 241 United States TI 271 Colombia TI 291 United States TI 331Romania TI 380 Romania TI 381 United States TI 395 United States TI 396United States TI 444 United States TI 480 United States TI 484 UnitedStates TI 486 United States TI 532 United States TI 538 Colombia TI 540Colombia TI 541 Honduras TI 567 Honduras TI 568 Ecuador TI 569 AlgeriaTI 69 Honduras TI 706 Iran TI 73 Venezuela TI 776 Former Soviet Union TI86 Former Soviet Union TI 87 Former Soviet Union TI 88 Former SovietUnion TI 90 Former Soviet Union TI 92 Former Soviet Union TI 93 FormerSoviet Union TI 94 Brazil TI 97 Brazil TI 975 TI 1007 TI 1025 TI 1026Tabaco Corriente TI 105 Ambireno TI 1050 Cuba TI 1061 Lampazo TI 1067Hoja Parado (Galpao) TI 1068 Judi Pride Bertel TI 1075 AmericanoTracuateua TI 108 Guayabito TI 1080 CrIDo Saltono TI 1082 CrIDo SaltonoTI 1083 Chileno Colorado, Hoja Anjosta TI 1085 Chileno Grande ColoradoTI 1095 Creja De Mula TI 1119 Chinese X Amarellinho TI 1143 Cubano De LaSierra TI 115 TI 119 TI 1211 TI 1215 TI 1277 TI 1288 Begej TI 1331 FodyaTI 1350 TI 1352 Oxviz TI 1356 Kulsko TI 1380 Tekne TI 1388 Nuk TI 1397AmarIDo Rio Grande Do Sul TI 14 Guacharo U.S.A. TI 1473 TI 1482 TI 1484Rippel TI 1498 Amarelao TI 1499 Immune 580 MS TI 1501 W.K. 39 TI 1502Sirone TI 1508 Espado TI 151 Simmaba TI 152 Russian Burley TI 1534Vorstenladen TI 1541 Selesion Olor TI 1543 NF 2617 TI 1550 NFC2 TI 1551Kutsaga E-1 TI 1552 CH T.Z. 273-3B TI 1556 Beinhart 1000-1 TI 1561Lonibow TI 1573 A17 TI 1574 A22 TI 1575 A23 TI 1576 Parado TI 1583 QuinDiaz TI 1585 Ke-Shin No. 1 TI 1592 BT 101 TI 1594 Shiroenshu 201 TI 1604Higo TI 161 Lonibow TI 1613 Little Gold 1025 TI 1618 MA-Song-Ta TI 1619Nanbu TI 162 Tan-Yuh-1 TI 1620 Veliki Hercegovac TI 1623 (S.P.I. 27525)TI 178 Cordoba TI 198 Virginia TI 220 Virginia TI 222 No. 3 TI 230Cordoba TI 255 Cordoba TI 257 Cordoba TI 260 Cordoba TI 268 Cubano TI295 TI 301 HojaAncha TI 309 TI 312 Chocoa TI 313 Palmira TI 318 CubanoTI 323 TI 341 TI 343 TI 350 TI 382 Zapatoca TI 384 Tachuleo TI 385Arcial Chico TI 394 TI 407 Copan TI 421 Virginia TI 424 TI 429 TachueloTI 432 CordoncIDo TI 438 Repello and Bravo Negro TI 445 TI 447 CostIDoNigro, Blanco, Pina TI 450 Hubana and Palmira TI 476 Colorado TI 508 TI510 TI 514 Chaco Chivo TI 515 Kentucky TI 527 TI 528 Tabaco Blanco TI530 TI 554 Cacho Do Chivo TI 560 Dolores De Copan TI 562 Barbasco TI 578TI 582 TI 592 TI 596 TI 606 TI 629 Blanco, Colorado TI 630 Tlapacoyan TI645 TI 657 TI 661 Oia-De-Vastago TI 665 Chanchamayo TI 687 Daule TI 691TI 717 AmarIDo Riogrande TI 74 Monte Libano TI 764 TI 785 Virginia TI789 TI 792 Cacerio De Songoy TI 794 Gumo TI 797 TI 822 Negro or Salom TI870 Capadare and Rabo De Gallo TI 889 TI 946 Rabo De Gallo TI 955Virginia Bright TI 964 KY171 (ph) 04GH#105-1 KY171 (ph) 04GH#105-2 KY171(ph) 04GH#105-3 KY171 (ph) 04GH#105-4 KYI71 (ph_) 04GH#105-5 KY171 (ph)04GH#105-6 KY171 (ph) 04GH#107-1 KY171 (ph) 04GH#107-2 KY171 (ph)04GH#107-3 KY171 (ph) 04GH#107-4 KY171 (ph) 04GH#107-5 KY171 (ph)04GH#107-6 NL. Madole (ph) 04GH#114-1 NL. Madole (ph) 04GH#114-2 NL.Madole (ph) 04GH#114-3 NL. Madole (ph_) 04GH#114-4 NL. Madole (ph)04GH#114-5 NL. Madole (ph) 04GH#114-6 NL. Madole (ph) 04GH#115-1 NL.Madole (ph) 04GH#115-2 NL. Madole (ph) 04GH#115-3 NL. Madole (ph)04GH#115-4 NL. Madole (ph) 04GH#115-5 NL. Madole (ph) 04GH#115-6 TN D950(ph_) 04GH#124-1 TN D950 (ph) 04GH#124-2 TN D950 (ph) 04GH#124-3 TN D950(ph) 04GH#124-4 TN D950 (ph) 04GH#124-5 TN D950 (ph) 04GH#124-6 TN D950(ph) 04GH#125-1 TN D950 (ph) 04GH#125-2 TN D950 (ph) 04GH#125-3 TN D950(ph) 04GH#125-4 TN D950 (ph) 04GH#125-5 TN D950 (ph) 04GH#125-6 Basma(PhPh) 04GH#68 KY14 86-00-K-7-1

Leaf samples were taken from six-week-old plants. DNA extractions fromthe leaves were performed using DNeasy Plant Mini Kit (Qiagen, Inc.,Valencia, Calif.) according to manufacturer's protocol.

The primers were designed based on the 5′ promoter and 3′ UTR regionsdescribed herein. The forward primer was 5′-GGC TCT AGA TAA ATC TCT TAAGTTACT AGG TIC TAA-3′ (SEQ NO: 2290) and the reverse primer was 5′-GGCTCT AGA AGT CAA TTA TCT TCT ACA AAC CTT TAT ATA TTA GC-3′ (SEQ ID NO:2291) (from −750 of the 5′ flanking region to 180 nt 3′ UTR). GenomicDNA extracted from all above-mentioned Nicotiana lines was used for thePCR analysis. A 100,ID reaction mixture and the Pfx high fidelity enzymewere used for PCR amplification. The annealing temperature used was 54°C. due to less homology among the species (this temperature is 2° C.lower than the temperature used for cloning genomic sequence from 4407converter tobacco as described above). The PCR product was visualized on0.8% agarose gel after electrophoresis A single band with molecularweight of approximately 3.5 kb was either present or absent on the gel.The lines with a positive band were scored as having the target gene.For the lines that lacked positive bands, four additional PCR reactionswere performed using four more sets of primers. These sets of primerswere selected from different regions of the gene. The four sets primerswere:

(1) from the start codon (SEQ ID NO: 2292)(5′- GCC CAT CCT ACA GTT ACC TAT AAA AAG GAA G3′) to the stop codon(SEQ ID NO: 2293) (5′- ACC AAG ATG AAA GAT CTT AGG TTT TAA -3′),(2) from 570 nt downstream of the start codon (SEQ ID NO: 2294)(5′- CTG ATC GTG AAG ATG A -3′) to the end of the intron(SEQ ID NO: 2295) (5′- TGC TGC ATC CAA GAC CA -3′),(3) from 300 nt downstream of the beginning of the intron(SEQ ID NO: 2296) (5′- GGG CTA TAT GGA TTC GC -3′)to the end of the intron (SEQ ID NO: 2295)(5′- TGCTGC ATC CAA GAC CA -3′), and(4) from 300 nt downstream of the beginning of the intron(SEQ ID NO: 2296) (5′- GGG CTA TAT GGA TTC GC -3′) to the 3' UTR(SEQ ID NO: 2195) (5′- AGT CAA TTA TCT TCT ACA AAC CTT TAT ATA TTAGC -3′).

If the five above-mentioned PCR reactions all showed no correct bands,the line was scored as lacking the target gene. Examples of the genomicDNA quantity and PCR products for the target nicotine demethylase geneare depicted in FIGS. 2 and 3.

Germplasm identified as lacking the nicotine demethylase gene is used assource material for breeding with cultivated tobaccos. However, anynucleic acid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193,or a fragment thereof, can be used in a similar manner. Interspecific orintraspecific hybridization methods combined with standard breedingmethods, such as backcrossing or the pedigree method, may be used totransfer the aberrant or absent nicotine demethylase gene or any nucleicacid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, or afragment thereof, from the donor source to cultivated tobaccos. Resultsof screening experiments for nicotine demethylase are set forth in Table9 below. A line negative for nicotine demethylase may be bred withitself or another negative line (e.g., Nicotiana africana×Nicotianaafricana or Nicotiana africana×Nicotiana amplexicaulis or any suitablebreeding combination). Negative lines are also bred with any commercialvariety of tobacco according to standard tobacco breeding techniquesknown in the art. Tobacco lines may be bred with any other compatibleplant according to standard procedures in the art.

TABLE 9 Exemplary Results from Screening the Nicotiana Genus for theNicotine Demethylase Gene Scientific Name or Common Name or (Origin)Inventory Number Screening Results Nicotiana africana TW6 NegativeNicotiana amplexicaulis TW10 Negative Nicotiana arentsii TW12 NegativeNicotiana benthamiana TW16 Negative Nicotiana bigelovii TW18 NegativeNicotiana corymbosa TW35 Negative Nicotiana debneyi TW36 NegativeNicotiana excelsior TW46 Negative Nicotiana exigua TW48 NegativeNicotiana glutinosa TW58 Negative Nicotiana goodspeedii TW67 NegativeNicotiana gossei TW68 Negative Nicotiana hesperis TW69 NegativeNicotiana ingulba TW71 Negative Nicotiana knightiana TW73 NegativeNicotiana maritima TW82 Negative Nicotiana megalosiphon TW83 NegativeNicotiana miersii TW85 Negative Nicotiana nesophila TW87 NegativeNicotiana noctiflora TW88 Negative Nicotiana nudicaulis TW90 NegativeNicotiana otophora TW94 Positive Nicotiana palmeri TW98 NegativeNicotiana paniculata TW99 Negative Nicotiana petunioides TW105 NegativeNicotiana plumbaginifolia TW106 Negative Nicotiana repanda TW110Negative Nicotiana rosulata TW112 Negative Nicotiana rotundifolia TW114Negative Nicotiana rustica TW116 Negative Nicotiana setchelli TW121Negative Nicotiana stocktonii TW126 Negative Nicotiana eastii TW127Negative Nicotiana suaveolens TW128 Negative Nicotiana thrysiflora TW139Positive Nicotiana tomentosa TW140 Positive Nicotiana tomentosiformisTW142 Positive Nicotiana trigonophylla TW143 Negative NLMadoleFoundation seed Positive KY 14 Foundation seed Positive TN 86 Foundationseed Positive Coker 176 Foundation seed Positive KY21 TC62 Positive KY22TC63 Positive KY24 TC64 Positive KY26 TC65 Positive KY33 TC66 PositiveKY34 TC67 Positive KY35 TC68 Positive KY41A TC69 Positive KY54 TC71Positive KY52 TC70 Positive Virginia 528 TC85 Positive Virginia B-29TC86 Positive 401 Cherry Red TC227 Positive 401 Cherry Red Free TC228Positive KY170 TC474 Positive KY171 TC475 Positive Maryland 609 TC505Positive Maryland Mammoth TC507 Positive VA403 TC580 Positive KY908TC630 Positive Earl Jennett Madole TC642 Positive Kavala TC533 PositiveKavala No 15A TC534 Positive GR 10 TC 19 Positive GR 10A TC20 PositiveGR24 TC27 Positive NOD 9 TI 1745 Positive NOD 12 TI 1747 Positive NOD 17TI 1749 Positive 80111 Pudawski 66CMS TI 1661 Positive 84160 Pudawski 66TI 1683 Positive MI1 109 TI 1715 Positive Mississippi Heirloom TI 1716Positive Ovens 62 TI 1741 Positive BT 101 TI 1594 Positive Kentucky MI429 TI 1595 Positive Shiroenshu 201 TI 1604 Positive Shiroenshu 202 TI1605 Positive Ostrolist2747 II TI 1568 Positive Ergo TI 1349 PositiveBurley 323 TI 1535 Positive Russian Burley TI 1534 Positive Puremozhetz83 TI 1569 Positive Bulsunov 80 TI 1537 Positive AmarIDo Riogrande TI74Positive Espado TI151 Positive CrIDo Saltono TI1082 Positive Kutsaga E-1TI1552 Positive Beinhart 1000-1 TI1561 Positive Kelly Brownleaf TC50Positive KY9 TC54 Positive Black Mammoth TC460 Positive Lizard TailOrinoco TC477 Positive Bel MS-2 TC493 Positive Maryland 201 TC503Positive Perique TC556 Positive NC-BMR 90 TC571 Positive LN KY 171 TC60SPositive Samsun TC536 Positive Xanthi-Parental TC554 Positive (Turkey)TI 1222 Positive Hongrois (Spain) TI 1246 Positive (Ethiopia) TI 1269Positive Ravajk(Yugoslavia) TI 1284 Positive (Bolivia) TI 1301 PositiveAdjuctifolia (New Zealand) TI 1317 Positive NO. 6055 (Cuba) TI 1375Positive (Bulgaria) TI 1386 Positive Grande Reditto (Italy) TI 1414Positive (Germany) TI 1459 Positive (Switzerland) TI 1506 PositiveSirone (Australia) TI 1508 Positive Dubek 566 (Poland) TI 1567 PositiveKagoshima Mamba (Japan) TI 158 Positive Erzegovina Lecce MI 411(Italy)TI 1602 Positive (Colombia) TI 291 Positive Okso (Former Soviet Union)TI 86 Positive

Example 18 Creating or Generating Mutations and Screening for GeneticVariation in the Nicotine Demethylase Gene

Preexisting genetic variation or mutations in the sequence coding forthe nicotine demethylase or any other genes represented by a nucleicacid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, or afragment thereof, are screened using molecular technologies includingtargeted induced local lesions in genomes (TIDING), DNA fingerprintingmethods such as amplified fragment length polymorphisms (AFLP), andsingle nucleotide polymorphisms (SNP). In practice, plant populationsrepresenting preexisting genetic variation such as a transgenic plant(e.g., any of those described herein) or those created by exposingreproductive tissues, seed, or other plant tissues to chemical mutagenssuch as alkylating agents, ethane methyl sulfonate (EMS) for example, orto radiation such as x-rays or gamma rays are used. For mutagenizedpopulations the dosage of the mutagenic chemical or radiation isdetermined experimentally for each type of plant tissue such that amutation frequency is obtained that is below a threshold levelcharacterized by lethality or reproductive sterility. The number of M1generation seed or the size of M1 plant populations resulting from themutagenic treatments are estimated based upon the expected frequency ofmutations. The progeny, M2 generation, of the M1 plants represent thepopulation that desirably is evaluated for a mutation in a gene, e.g.,the nicotine demethylase gene.

Tilling, DNA fingerprinting, SNP or similar technologies may be used todetect induced or naturally-occurring genetic variation in a desirablegene such as the nicotine demethylase gene. The variation may resultfrom deletions, substitutions, point mutations, translocations,inversions, duplications, insertions or complete null mutations. Thesetechnologies could be used in a marker-assisted selection (MA breedingprogram) to transfer or breed the null or dissimilar alleles of thenicotine demthylase gene or any nucleic acid sequence shown in FIGS. 2to 7 and SEQ ID NOS: 446 to 2193, or a fragment thereof, into othertobaccos. A breeder could create segregating populations fromhybridizations of a genotype containing the null or dissimilar allelewith an agronomically desirable genotype. Plants in the F2 or backcrossgenerations could be screened using a marker developed from the nicotinedemethylase sequence or a nucleic acid sequence shown in FIGS. 2 to 7and SEQ ID NOS: 446 to 2193, or a fragment thereof, using one of thetechniques listed previously. Plants identified as possessing the nullor dissimilar alleles could be backcrossed or self-pollinated to createthe next population that could be screened. Depending on the expectedinheritance pattern or the MAS technology used, it may be necessary toself-pollinate the selected plants before each cycle of backcrossing toaid identification of the desired individual plants. Backcrossing orother breeding procedure can be repeated until the desired phenotype ofthe recurrent parent is recovered.

Example 19 Breeding or Transfer of Variant Nictoine Demethylase GeneExpression into Cultivated Tobacco A. Selection of Parental Lines

Donor tobacco lines are identified as those having variant nicotinedemethylase gene expression (e.g., a tobacco line identified using aPCR-based strategy as lacking the nicotine demethylase gene or is nullfor nicotine demethylase or expressing a nicotine demethylase havingaltered enzymatic activity; or a tobacco line expressing a trailsgenethat alters or silences gene expression is also considered to be variantfor nicotine demethylase gene expression) or variants of any nucleicacid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 21934, or afragment thereof, and are selected to serve as the donor parent. Suchplants are generated according to standard methods known in the art,e.g., those described herein. Other donor plants include tobacco plantsthat have been mutagenized and subsequently identified as having variantnicotine demethylase gene activity or variant activity of a gene productencoded by any nucleic acid sequence shown in FIGS. 2 to 7 and SEQ IDNOS: 446 to 2193, or a fragment thereof. One exemplary donor parent isthe tobacco line, Nicotiana rustica.

The recipient tobacco line is typically any commercial tobacco varietysuch as Nicotiana tabacum TN 90. Other useful Nicotiana tabacumvarieties include BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500,CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold,Coker 48, CU263, DF911, Galpao tobacco, GL 26H, GL 350, GL 737, GL 939,GL 973, HB 04P, K 149, K 326, K 346, K 358, K 394, K 399, K 730, KT 200,KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY 160, Little Crittenden,McNair 373, McNair 944, msKY 14×L8, Narrow Leaf Madole, NC 100, NC 102,NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC 606, NC 71,NC 72, NC 810, NC BH 129, OXFORD 207, ‘Perique’ tobacco, PVH03, PVH09,PVHI9, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H4,RGH51, RGH 4, RGH 51, RS 1410, SP 168, SP 172, SP 179, SP 210, SP 220,SP G-28, SP G-70, SP H20, SP NF3, TN 86, TN 97, TN D94, TN D950, TR (TomRosson) Madole, VA 309, VA 309, or VA 359. Seed from such varieties mayalso be from a source resulting from screening for the lack or presenceof nicotine conversion using standard chemical or molecular methods.Such commercial varieties also provide material for altering nicotinedemethylase activity according to the methods described herein. Othernull lines and recipient or donor lines known in the art are alsouseful, and lines identified as being dissimilar from the nicotinedemethylase gene described herein also serve as a donor parent.Recipient lines may also be chosen from any tobacco varieties forflue-cured, Burley, dark, Virginia or Oriental tobaccos.

Table 10 shows exemplary Nicotiana species which exhibit breedingcompatibility with Nicotiana tabacum (see also, for example, Compendiumof Tobacco Diseases published by APS or The Genus Nicotiana illustratedpublished by Japan Tobacco Inc.).

TABIE 10 Exemplary Nicotiana species compatible with Nicotiana tabacum.Scientific Name or Common Inventory Screening Name or (Origin) Number PINumber Results Nicotiana amplexicaulis TW10 PI 271989 Negative Nicotianabenthamiana TW16 PI 555478 Negative Nicotiana bigelovii TW18 PI 555485Negative Nicotiana debneyi TW36 Negative Nicotiana excelsior TW46 PI224063 Negative Nicotiana glutinosa TW58 PI 555507 Negative Nicotianagoodspeedii TW67 PI241012 Negative Nicotiana gossei TW68 PI 230953Negative Nicotiana hesperis TW69 PI 271991 Negative Nicotiana knightianaTW73 PI 555527 Negative Nicotiana maritima TW82 PI 555535 NegativeNicotiana megalosiphon TW83 PI 555536 Negative Nicotiana nudicaulis TW90PI 555540 Negative Nicotiana paniculata TW99 PI 555545 NegativeNicotiana plumbaginifolia TW106 PI 555548 Negative Nicotiana repandaTW110 PI 555552 Negative Nicotiana rustica TW116 Negative Nicotianasuaveolens TW128 PI 230960 Negative Nicotiana sylvestris TW136 PI 555569Negative Nicotiana tomentosa TW140 PI 266379 Positive Nicotiandtomentosiformis TW142 Positive Nicotiana trigonophylia TW143 PI 555572Negative

B. Gene Transfer

The donor parent is crossed or hybridized in a reciprocal manner withthe donor parent according to standard breeding methods. Successfulhybridizations, identified according to standard method, yield FI plantsthat are fertile or that are, if desired, backcrossed with the recipientparent. A plant population in the F2 generation, derived from the F1plant, is screened for variant nicotine demethylase gene expression(e.g., a plant is identified that fails to express nicotine demethylasedue to the absence of the nicotine demethylase gene according tostandard methods, for example, by using a peR method with primers basedupon the nucleotide sequence information for nicotine demethylasedescribed herein) or variant expression of any nucleic acid sequenceshown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, or a fragmentthereof. Alternatively, any standard screening method known in the artfor evaluating plant alkaloid content is used to identify plants that donot convert nicotine to nornicotine. Selected plants are then hybridizedwith the recipient parent and the first backcrossed (BCl) generationplants are self-pollinated to produce a BCIF2 population that is againscreened for variant nicotine demethylase gene expression (e.g., thenull version of the nicotine demethylase gene). The process ofbackcrossing, self-pollination, and screening is repeated, for example,at least 4 times until the final screening produces a plant that isfertile and reasonably similar to the recipient parent. This plant, ifdesired, is self-pollinated and the progeny are subsequently screenedagain to confirm that the plant exhibits variant nicotine demethylasegene expression (e.g., a plant that displays the null condition fornicotine demethylase) or variant expression of any nucleic acid sequenceshown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, or a fragmentthereof. Cytogenetic analyses of the selected plants is optionallyperformed to confirm the chromosome complement and chromosome pairingrelationships. Breeder's seed of the selected plant is produced usingstandard methods including, for example, field testing, confirmation ofthe null condition for nicotine demethylase or null or increasedcondition of a polypeptide encoded by any nucleic acid sequence shown inFIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, or a fragment thereof, andchemical analyses of cured leaf to determine the level of alkaloidsespecially the nornicotine content and the rationornicotine/nicotine+nornicotine or other such desired propertiesprovided by those gene sequences found in any nucleic acid sequenceshown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, or a fragmentthereof.

In situations where the original F1 hybrid resulting from the crossbetween the recipient (e.g., N. rustica) and donor parent (e.g., TN 90)is hybridized or backcrossed to the donor (e.g., TN 90), the progeny ofthis backcross is self-pollinated to create a BCIF2 generation that isscreened for the null or dissimilar version of nicotine demethylase orthe null or increased condition of a polypeptide encoded by any nucleicacid sequence shown in FIGS. 2 to 7 and SEQ ID NOS: 446 to 2193, or afragment thereof. The remainder of the breeding effort is as describedin the above paragraph.

C. Agronomic Performance Testing and Confirmation of Phenotype

Lines resulting from the breeding and screening for variant nicotinedemethylase gene expression (e.g., the null condition for nicotinedemethylase) or of any nucleic acid sequence shown in FIGS. 2 to 7 andSEQ ID NOS: 446 to 2193, or a fragment thereof, are evaluated in thefield using standard field procedures. Control genotypes including theoriginal recipient parent (e.g., TN 90) are included and entries arearranged in the filed in a randomized complete block design or otherappropriate field design. Standard agronomic practices for tobacco areused, for example, the tobacco is harvested, weighed, and sampled forchemical and other common testing before and during curing. Statisticalanalyses of the data are performed to confirm the similarity of theselected lines to the recipient, e.g., the parental line TN 90.

Example 20 Breeding or Transfer of a Modified Attribute Into CultivatedTobacco

Expression of any of the genes described herein, for example, anyone ofthose nucleic acid sequences shown in FIGS. 2 to 7 and SEQ ID NOS: 446to 2193 may also be modified according to the methods described herein.Such genes provide a basis for modifying a plant's phenotype, forexample, improving flavor or aroma or both, improving an organolepticproperty, or improving curability. Plants identified as having amodified phenotype are then used in a breeding protocol according tostandard methods known in the art, for example, those described herein.

Example 21 Hybrid Plant Generation

Application of standard protoplast culture methodologies developed forproduction of hybrid plants using protoplast fusion is also useful forgenerating plants having variant gene expression (e.g., variant nicotinedemethylase gene expression). Accordingly, protoplasts are generatedfrom a first and a second tobacco plant having variant gene expression.Calli are cultured from successful protoplast fusions and plants arethen regenerated. Resulting progeny hybrid plants are identified andselected for variant gene expression according to standard methods and,if desired, may be used in any standard breeding protocol.

WO 03/078577, WO 2004/035745, PCT/US/2004/034218, andPCT/US/2004/034065, and all other references, patents, patentapplication publications, and patent applications referred to herein areincorporated by reference herein to the same extent as if each of thesereferences, patents, patent application publications, and patentapplications were separately incorporated by reference herein.

Numerous modifications and variations in practice of the invention areexpected to occur to those skilled in the art upon consideration of theforegoing detailed description of the invention. Consequently, suchmodifications and variations are intended to be included within thescope of the following claims.

1. A breeding method for producing a tobacco plant having reducedconversion of nicotine to nornicotine, said method comprising the stepsof: (a) crossing at least one plant of a first tobacco line with atleast one plant of a second tobacco line, said plant of said firsttobacco line having an endogenous nucleic acid comprising a mutation inthe nucleotide sequence set forth in SEQ ID NO: 4; and (b) selectingprogeny of said cross that have said mutation, said progeny exhibitingreduced conversion of nicotine to nornicotine.
 2. The method of claim 1,wherein said mutation is selected from the group consisting of a pointmutation, a deletion, an insertion, a duplication and an inversion. 3.The method of claim 2, wherein said mutation is a point mutation.
 4. Themethod of claim 2, wherein said mutation is a deletion.
 5. The method ofclaim 1, wherein said selecting step comprises producing an F₂population and screening said F₂ population for said mutation.
 6. Themethod of claim 1, wherein said selecting step comprises producing abackcross population and screening said backcross population for saidmutation.
 7. The method of claim 6, wherein selecting step comprisesscreening F₃ progeny of said backcross population for said mutation. 8.The method of claim 6, wherein said selecting step comprises producing aBC₁F₂ population and screening said BC₁F₂ population for said mutation.9. The method of claim 8, wherein said selecting step comprisesbackcrossing, self-pollinating and screening progeny of a BC₁F₂population at least 4 times to obtain a plant that is fertile andexhibits reduced expression of said endogenous nucleic acid.
 10. Themethod of claim 1, further comprising the step of producing breeder'sseed, wherein plants produced from said seed have said mutation and saidreduced conversion of nicotine to nornicotine.
 11. The method of claim1, wherein said plant of said first tobacco line is an Oriental, a darktobacco, flue or air-cured tobacco, Virginia or a Burley tobacco plant.12. The method of claim 1, wherein said plant of said first tobacco lineis a Nicotiana tabacum plant.
 13. The method of claim 12, wherein saidplant of said first tobacco line is a dark tobacco plant.
 14. The methodof claim 1, wherein said plant of said second tobacco line is aNicotiana tabacum plant.
 15. The method of claim 1, wherein said plantof said first tobacco line is a male sterile plant or a male sterilehybrid plant.
 16. A method of breeding a tobacco plant, said methodcomprising the steps of: a) crossing a plant of a first tobacco linewith a plant of a second tobacco line to produce F₁ progeny tobaccoplants, said first tobacco plant having an endogenous nucleic acidcomprising a mutation in the nucleotide sequence set forth in SEQ ID NO:4; b) extracting a DNA sample from each of a plurality of said F₁progeny tobacco plants; c) contacting each said DNA sample with a markernucleic acid that hybridizes to said endogenous nucleic acid or to afragment thereof; and d) performing a marker assisted breeding method toproduce a progeny tobacco line, said progeny tobacco line having saidmutation and exhibiting reduced conversion of nicotine to nornicotine.17. The method of claim 16, wherein said performing step comprisesutilizing a molecular technology selected from the group consisting ofamplified fragment length polymorphism, restriction fragment lengthpolymorphism, random amplified polymorphism display, single nucleotidepolymorphism, a microsatellite marker, or targeted induced local lesionin a tobacco genome.
 18. A method of producing tobacco seed, comprisinga) crossing a plant of a first tobacco line with a plant of a secondtobacco line, said first tobacco plant selected from the groupconsisting of Nicotiana africana, Nicotiana amplexicaulis, Nicotianaarentsii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotianacorymbosa, Nicotiana debneyi, Nicotiana excelsior, Nicotiana exigua,Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotianahesperis, Nicotiana ingulba, Nicotiana knightiana, Nicotiana maritima,Nicotiana megalosiphon, Nicotiana miersii, Nicotiana nesophila,Nicotiana noctiflora, Nicotiana nudicaulis, Nicotiana otophora,Nicotiana palmeri, Nicotiana paniculata, Nicotiana petunioides,Nicotiana plumbaginifolia, Nicotiana repanda, Nicotiana rosulata,Nicotiana rotundifolia, Nicotiana rustica, Nicotiana setchelli,Nicotiana stocktonii, Nicotiana eastii, and Nicotiana suaveolens, andsaid second tobacco plant having an endogenous nucleic acid comprising amutation in the nucleotide sequence set forth in SEQ ID NO: 4; and b)harvesting seed produced from said cross.
 19. The method of claim 18,wherein said second tobacco plant is Nicotiana tabacum.
 20. The methodof claim 18, wherein said first tobacco plant is Nicotiana rustica.